Methods of using tumor infiltrating lymphocytes in double-refractory melanoma

ABSTRACT

Methods of treating melanomas refractory to other therapies using tumor infiltrating lymphocytes are disclosed. Also disclosed is the use of IP-10 as a biomarker for predicting treatment efficacy.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation in part of International PatentApplication No. PCT/US2018/036088, filed Jun. 5, 2018, which claims thebenefit of U.S. Provisional Patent Application No. 62/515,257, filedJun. 5, 2017, each of which is incorporated by reference in itsentirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 5, 2018, isnamed 116983-5047_ST25.txt and is 122 kilobytes in size.

FIELD OF THE INVENTION

Methods of using tumor infiltrating lymphocytes (TILs) in the treatmentof double-refractory melanoma are disclosed herein, as well as the useof IP-10 as a biomarker for predicting treatment efficacy.

BACKGROUND OF THE INVENTION

Treatment of melanoma remains challenging, particularly for patientsthat do not respond to commonly-used initial lines of therapy, includingnivolumab monotherapy, pembrolizumab monotherapy, therapy using acombination of nivolumab and ipilimumab, ipilimumab monotherapy, therapyusing a combination of dabrafenib and trametinib, vemurafenibmonotherapy, and pegylated interferon (preinterferon) alfa-2b. Approvedfirst line treatments for metastatic melanoma include immunotherapeuticstrategies blocking PD-1 (pembrolizumab, nivolumab), or combiningnivolumab with the anti-CTLA4 blocker ipilimumab, or chemotherapy withagents targeting specific activating mutations in the BRAF pathway(e.g., vemurafenib, dabrafenib, trametinib). Following diseaseprogression, patients can receive additional treatment with anti-PD-1monotherapy; nivolumab/ipilimumab combination therapy; ipilimumabmonotherapy; targeted therapy if BRAF mutant; high-dose aldesleukin(interleukin-2; IL-2); cytotoxic agents (e.g., dacarbazine,temozolomide, paclitaxel, cisplatin, carboplatin, vinblastine); orimatinib for KIT-mutant melanoma. In 2015, talimogene laherparepvec, alive oncolytic virus therapy, was approved for the local treatment ofunresectable cutaneous, subcutaneous, and nodal lesions in patients withmelanoma recurrent after initial surgical excision. This product has notbeen shown to improve overall survival or to have an effect on visceralmetastases.

Until recently, high-dose aldesleukin was the only FDA-approved systemictherapy for metastatic melanoma capable of inducing durable objectivecancer responses, with an overall objective response rate (ORR) of 16%and durable complete tumor regressions (CRs) observed in up to 6% oftreated patients (Proleukin® (aldesleukin) Label, FDA, July 2012). Alva,et al. Cancer Immunol. Immunother. 2016, 65, 1533-1544. The recentlyapproved PD-1 immune checkpoint inhibitors pembrolizumab and nivolumabapproximately double the rate of durable responses in metastaticmelanoma relative to aldesleukin treatment. Larkin, et al., N. Engl. J.Med. 2015, 373, 23-34; Robert, et al., N. Engl. J. Med. 2015, 372,2521-32. In previously treated patients, the ORR for nivolumab is 32%,with higher and more durable responses correlated with higher levels ofPD-1 ligand expression by tumors; and the ORR for pembrolizumabfollowing prior therapy with ipilimumab is 21% (Table 2). In treatmentnaïve patients, durable objective responses are achieved in 50% ofpatients when nivolumab and ipilimumab administered in combination,although the CR rate remains low at 8.9% (Opdivo® (nivolumab) Label,FDA, October 2016).

Use of the checkpoint inhibitors is associated with a spectrum ofimmune-related adverse events, including pneumonitis, colitis,hepatitis, nephritis and renal dysfunction (Opdivo (nivolumab) Label,FDA, October 2016). Hofmann, et al., Eur. J. Cancer 2016, 60, 190-209.Increased toxicity is observed in patients treated with nivolumab andipilimumab combination therapy. Treatment-related adverse events leadingto discontinuation of therapy occurred in 36.4%, 7.7% and 14.8% ofpatients receiving the combination therapy, nivolumab alone oripilimumab alone, respectively. Larkin, et al., N. Engl. J. Med. 2015,373, 23-34; Johnson, et al., N. Engl. J. Med. 2016, 375, 1749-1755.

Although the targeted therapies and immune checkpoint inhibitors canachieve dramatic responses in patients with metastatic melanoma, deathrates for this cancer are projected to remain stable through 2030. Theoverall age-adjusted melanoma death rate was 2.7 per 100000 in 2011 andremained at this level in 2015. Guy, et al., Morbidity Mortality WeeklyRep. 2015, 64, 591-596.

Treatment of bulky, refractory cancers using adoptive autologoustransfer of tumor infiltrating lymphocytes (TILs) represents a powerfulapproach to therapy for patients with poor prognoses. Gattinoni, et al.,Nat. Rev. Immunol. 2006, 6, 383-393. TILs are dominated by T cells, andIL-2-based TIL expansion followed by a “rapid expansion process” (REP)has become a preferred method for TIL expansion because of its speed andefficiency. Dudley, et al., Science 2002, 298, 850-54; Dudley, et al.,J. Clin. Oncol. 2005, 23, 2346-57; Dudley, et al., J. Clin. Oncol. 2008,26, 5233-39; Riddell, et al., Science 1992, 257, 238-41; Dudley, et al.,J. Immunother. 2003, 26, 332-42. A number of approaches to improveresponses to TIL therapy in melanoma and to expand TIL therapy to othertumor types have been explored with limited success, and the fieldremains challenging. Goff, et al., J. Clin. Oncol. 2016, 34, 2389-97;Dudley, et al., J. Clin. Oncol. 2008, 26, 5233-39; Rosenberg, et al.,Clin. Cancer Res. 2011, 17, 4550-57. There is an unmet need tostandardize TIL production as well as identify patient populations andspecific types of cancers that are most likely to benefit from TILtherapy.

The present invention provides the surprising finding that TILs may beused in the treatment of a subpopulation of patients suffering frommelanoma that is refractory to at least two prior therapies, which mayinclude immune checkpoint inhibitors. Also disclosed is the use of IP-10as a biomarker for predicting treatment efficacy.

SUMMARY OF THE INVENTION

In an embodiment, the present disclosure provides a method of treatingdouble-refractory metastatic melanoma in a patient in need thereof, themethod comprising administering a therapeutically effective populationof tumor infiltrating lymphocytes (TILs) to the patient.

In an embodiment and in accordance with the above, wherein thedouble-refractory metastatic melanoma is a cutaneous double-refractorymetastatic melanoma.

In an embodiment and in accordance with any of the above, thedouble-refractory metastatic melanoma is refractory to at least twoprior systemic treatment courses, not including neo-adjuvant or adjuvanttherapies.

In an embodiment and in accordance with any of the above, thedouble-refractory metastatic melanoma is refractory to aldesleukin or abiosimilar thereof.

In an embodiment and in accordance with any of the above, thedouble-refractory metastatic melanoma is refractory to pembrolizumab ora biosimilar thereof.

In an embodiment and in accordance with any of the above, thedouble-refractory metastatic melanoma is refractory to nivolumab or abiosimilar thereof.

In an embodiment and in accordance with any of the above, thedouble-refractory metastatic melanoma is refractory to ipilimumab or abiosimilar thereof.

In an embodiment and in accordance with any of the above, thedouble-refractory metastatic melanoma is refractory to ipilimumab or abiosimilar thereof and pembrolizumab or a biosimilar thereof.

In an embodiment and in accordance with any of the above, thedouble-refractory metastatic melanoma is refractory to ipilimumab or abiosimilar thereof and nivolumab or a biosimilar thereof.

In an embodiment and in accordance with any of the above, thedouble-refractory metastatic melanoma is refractory to a BRAF inhibitor.

In an embodiment and in accordance with any of the above, thedouble-refractory metastatic melanoma is refractory to a PD-L1inhibitor.

In an embodiment and in accordance with any of the above, the PD-L1inhibitor is selected from the group consisting of avelumab,atezolizumab, durvalumab, and biosimilars thereof.

In an embodiment and in accordance with any of the above, thedouble-refractory metastatic melanoma is refractory to a combination ofa PD-1 inhibitor and a CTLA-4 inhibitor.

In an embodiment and in accordance with any of the above, the PD-1inhibitor is nivolumab or a biosimilar thereof and the CTLA-4 inhibitoris selected from the group consisting of ipilumumab, tremelimumab, andbiosimilars thereof.

The In an embodiment and in accordance with any of the above, thedouble-refractory metastatic melanoma is refractory to a combination ofa BRAF inhibitor and a MEK inhibitor.

In an embodiment and in accordance with any of the above, the BRAFinhibitor is dabrafenib or a pharmaceutically-acceptable salt thereofand the MEK inhibitor is trametinib or a pharmaceutically-acceptablesalt or solvate thereof.

In an embodiment and in accordance with any of the above, the metastaticmelanoma is resistant to a PD-1 inhibitor or PD-L1 inhibitor.

In an embodiment and in accordance with any of the above, the PD-1 orPD-L1 inhibitor is selected from the group consisting of nivolumab,pembrolizumab, avelumab, atezolizumab, durvalumab, and biosimilarsthereof.

In an embodiment and in accordance with any of the above, the patientdoes not possess a BRAF mutation.

In an embodiment and in accordance with any of the above, the patienthas received at most 4 doses of nivolumab or a biosimilar thereof priorto receiving the therapeutically effective population of TILs.

In an embodiment and in accordance with any of the above, the patienthas progressed or had no response to at least two prior systemictreatment courses.

In an embodiment and in accordance with any of the above, the patientexhibits an increase in the level of IP-10 after administration of thetherapeutically effective population of tumor infiltrating lymphocytes(TILs).

In an embodiment and in accordance with any of the above, the increasein the level of IP-10 is indicative of treatment response and/ortreatment efficacy.

In an embodiment and in accordance with any of the above, the increasein the level of IP-10 is measured by calculating the difference in IP-10level in plasma seven days before TIL infusion and one day after TILinfusion, and wherein said difference in IP-10 level in plasma is atleast 800 pg/mL, at least 900 pg/mL, at least 1000 pg/mL, at least 1100pg/mL, at least 1200 pg/mL, at least 1300 pg/mL, at least 1400 pg/mL, atleast 1500 pg/mL, at least 1600 pg/mL, at least 1650 pg/mL, at least1656 pg/mL, at least 1700 pg/mL, or at least 1800 pg/mL, at least 1900pg/mL, at least 2000 pg/mL, at least 2100 pg/mL, or at least 2200 pg/mL.

In an embodiment and in accordance with any of the above, the patient isadministered one or more further dosages of a therapeutically effectivepopulation of tumor infiltrating lymphocytes (TILs).

In an embodiment and in accordance with any of the above, the patient isnot administered a further dosage of a therapeutically effectivepopulation of tumor infiltrating lymphocytes (TILs).

In an embodiment, the present disclosure provides method of treatingdouble-refractory metastatic melanoma in a patient in need thereof, themethod comprising:

-   -   (a) obtaining a first population of TILs from a tumor resected        from the patient by processing a tumor sample obtained from the        patient into multiple tumor fragments;    -   (b) adding the tumor fragments into a closed system;    -   (c) performing a first expansion by culturing the first        population of TILs in a cell culture medium comprising IL-2, and        optionally OKT-3, to produce a second population of TILs,        wherein the first expansion is performed in a closed container        providing a first gas-permeable surface area, wherein the first        expansion is performed for about 3-14 days to obtain the second        population of TILs, wherein the second population of TILs is at        least 50-fold greater in number than the first population of        TILs, and wherein the transition from step (b) to step (c)        occurs without opening the system;    -   (d) performing a second expansion by supplementing the cell        culture medium of the second population of TILs with additional        IL-2, optionally OKT-3, and antigen presenting cells (APCs), to        produce a third population of TILs, wherein the second expansion        is performed for about 7-14 days to obtain the third population        of TILs, wherein the third population of TILs is a therapeutic        population of TILs, wherein the second expansion is performed in        a closed container providing a second gas-permeable surface        area, and wherein the transition from step (c) to step (d)        occurs without opening the system;    -   (e) harvesting the therapeutic population of TILs obtained from        step (d) to provide a harvested TIL population, wherein the        transition from step (d) to step (e) occurs without opening the        system;    -   transferring the harvested TIL population from step (e) to an        infusion bag, wherein the transfer from step (e) to (f) occurs        without opening the system, and optionally cryopreserving the        harvested TIL population and    -   (g) administering a therapeutically effective amount of the        harvested TIL population to the patient with double-refractory        metastatic melanoma.

In an embodiment and in accordance with the above-described method,wherein the patient has been previously treated with a PD-1 inhibitor ora biosimilar thereof.

In an embodiment and in accordance with any of the above, wherein thePD-1 inhibitor is selected from the group consisting of nivolumab,pembrolizumab, and biosimilars thereof.

In an embodiment and in accordance with any of the above, wherein thepatient has been previously treated with a PD-L1 inhibitor or abiosimilar thereof.

In an embodiment and in accordance with any of the above, wherein thePD-L1 inhibitor is selected from the group consisting of avelumab,atezolizumab, durvalumab, and biosimilars thereof.

In an embodiment and in accordance with any of the above, wherein thePD-1 inhibitor or a biosimilar thereof was co-administered with a CTLA-4inhibitor or biosimilar thereof.

In an embodiment and in accordance with any of the above, wherein thePD-L1 inhibitor or a biosimilar thereof was co-administered with aCTLA-4 inhibitor or biosimilar thereof.

In an embodiment and in accordance with any of the above, wherein thepatient has been previously treated with one additional prior line ofsystemic therapy.

In an embodiment and in accordance with any of the above, wherein theone additional prior line of systemic therapy is a BRAF inhibitor or apharmaceutically-acceptable salt thereof.

In an embodiment and in accordance with any of the above, wherein theBRAF inhibitor is selected from the group consisting of vemurafenib,dabrafenib, and pharmaceutically-acceptable salts thereof.

In an embodiment and in accordance with any of the above, wherein theone additional prior line of systemic therapy is a MEK inhibitor or apharmaceutically-acceptable salt or solvate thereof.

In an embodiment and in accordance with any of the above, wherein theMEK inhibitor is selected from the group consisting of trametinib,cobimetinib, and pharmaceutically-acceptable salts or solvates thereof.

In an embodiment and in accordance with any of the above, wherein theone additional prior line of systemic therapy is a combination of a BRAFinhibitor or a pharmaceutically-acceptable salt thereof and a MEKinhibitor or a pharmaceutically-acceptable salt or solvate thereof.

In an embodiment and in accordance with any of the above, wherein theBRAF inhibitor is selected from the group consisting of vemurafenib,dabrafenib, and pharmaceutically-acceptable salts thereof, and the MEKinhibitor is selected from the group consisting of trametinib,cobimetinib, and pharmaceutically-acceptable salts or solvates thereof.

In an embodiment and in accordance with any of the above, wherein theone additional prior line of systemic therapy is a CTLA-4 inhibitor or abiosimilar thereof.

In an embodiment and in accordance with any of the above, wherein theCTLA-4 inhibitor is selected from the group consisting of ipilumumab,tremelimumab, and biosimilars thereof.

In an embodiment and in accordance with any of the above, wherein theone additional prior line of systemic therapy is chemotherapeuticregimen.

In an embodiment and in accordance with any of the above, wherein thechemotherapeutic regimen comprises dacarbazine or temozolimide.

In an embodiment and in accordance with any of the above, wherein thefirst expansion is performed over a period of about 11 days.

In an embodiment and in accordance with any of the above, wherein theIL-2 is present at an initial concentration of between 1000 IU/mL and6000 IU/mL in the cell culture medium in the first expansion step (c).

In an embodiment and in accordance with any of the above, wherein in thesecond expansion step (d), the IL-2 is present at an initialconcentration of between 1000 IU/mL and 6000 IU/mL and the OKT-3antibody is present at an initial concentration of about 30 ng/mL.

In an embodiment and in accordance with any of the above, wherein thefirst expansion is performed using a gas permeable container.

In an embodiment and in accordance with any of the above, wherein thesecond expansion is performed using a gas permeable container.

In an embodiment and in accordance with any of the above, wherein thecell culture medium in the first expansion step (c) further comprises acytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21,and combinations thereof.

In an embodiment and in accordance with any of the above, wherein thecell culture medium in the second expansion step (d) further comprises acytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21,and combinations thereof.

In an embodiment and in accordance with any of the above, furthercomprising the step of treating the patient with a non-myeloablativelymphodepletion regimen prior to administering the TILs to the patient.

In an embodiment and in accordance with any of the above, wherein thenon-myeloablative lymphodepletion regimen comprises the steps ofadministration of cyclophosphamide at a dose of 60 mg/m2/day for twodays followed by administration of fludarabine at a dose of 25 mg/m2/dayfor five days.

In an embodiment and in accordance with any of the above, furthercomprising the step of treating the patient with an IL-2 regimenstarting on the day after administration of the TILs to the patient.

In an embodiment and in accordance with any of the above, wherein theIL-2 regimen is a high-dose IL-2 regimen comprising 600,000 or 720,000IU/kg of aldesleukin, or a biosimilar or variant thereof, administeredas a 15-minute bolus intravenous infusion every eight hours untiltolerance.

In an embodiment and in accordance with any of the above, the patientexhibits an increase in the level of IP-10 after administration of thetherapeutically effective population of tumor infiltrating lymphocytes(TILs).

In an embodiment and in accordance with any of the above, the increasein the level of IP-10 is measured by calculating the difference in IP-10level in plasma seven days before TIL infusion and one day after TILinfusion, and wherein said difference in IP-10 level in plasma is atleast 800 pg/mL, 900 pg/mL, at least 1000 pg/mL, at least 1100 pg/mL, atleast 1200 pg/mL, at least 1300 pg/mL, at least 1400 pg/mL, at least1500 pg/mL, at least 1600 pg/mL, at least 1650 pg/mL, at least 1656pg/mL, at least 1700 pg/mL, or at least 1800 pg/mL, at least 1900 pg/mL,at least 2000 pg/mL, at least 2100 pg/mL, or at least 2200 pg/mL.

In an embodiment and in accordance with any of the above, the increasein the level of IP-10 is indicative of treatment efficacy.

In an embodiment and in accordance with any of the above, the patient isadministered one or more further dosages of a therapeutically effectivepopulation of tumor infiltrating lymphocytes (TILs).

In an embodiment and in accordance with any of the above, the patient isnot administered a further dosage of a therapeutically effectivepopulation of tumor infiltrating lymphocytes (TILs).

In an embodiment and in accordance with any of the above, wherein thetherapeutically effective population of TILs comprises from about2.3×1010 to about 13.7×1010 TILs.

In an embodiment, the present disclosure provides a method of treatingdouble-refractory metastatic melanoma in a patient in need thereof, themethod comprising:

-   -   (a) obtaining a first population of TILs from a tumor resected        from the patient by processing a tumor sample obtained from the        patient into multiple tumor fragments;    -   (b) adding the tumor fragments into a closed system;    -   (c) performing a first expansion by culturing the first        population of TILs in a cell culture medium comprising IL-2, and        optionally OKT-3, to produce a second population of TILs,        wherein the first expansion is performed in a closed container        providing a first gas-permeable surface area, wherein the first        expansion is performed for about 3-14 days to obtain the second        population of TILs, wherein the second population of TILs is at        least 50-fold greater in number than the first population of        TILs, and wherein the transition from step (b) to step (c)        occurs without opening the system;    -   (d) performing a second expansion by supplementing the cell        culture medium of the second population of TILs with additional        IL-2, optionally OKT-3, and antigen presenting cells (APCs), to        produce a third population of TILs, wherein the second expansion        is performed for about 7-14 days to obtain the third population        of TILs, wherein the third population of TILs is a therapeutic        population of TILs, wherein the second expansion is performed in        a closed container providing a second gas-permeable surface        area, and wherein the transition from step (c) to step (d)        occurs without opening the system;    -   (e) harvesting the therapeutic population of TILs obtained from        step (d) to provide a harvested TIL population, wherein the        transition from step (d) to step (e) occurs without opening the        system;    -   (f) transferring the harvested TIL population from step (e) to        an infusion bag, wherein the transfer from step (e) to (f)        occurs without opening the system, and optionally cryopreserving        the harvested TIL population;    -   (g) administering a therapeutically effective amount of the        harvested TIL population to the patient with double-refractory        metastatic melanoma; and    -   (h) measuring the level of IP-10 in the patient after        administering a therapeutically effective amount of the TILs in        step (g).

In an embodiment, the present disclosure provides a method of treatingcancer in a patient in need thereof, the method comprising:

-   -   (a) obtaining a first population of TILs from a tumor resected        from the patient by processing a tumor sample obtained from the        patient into multiple tumor fragments;    -   (b) adding the tumor fragments into a closed system;    -   (c) performing a first expansion by culturing the first        population of TILs in a cell culture medium comprising IL-2, and        optionally OKT-3, to produce a second population of TILs,        wherein the first expansion is performed in a closed container        providing a first gas-permeable surface area, wherein the first        expansion is performed for about 3-14 days to obtain the second        population of TILs, wherein the second population of TILs is at        least 50-fold greater in number than the first population of        TILs, and wherein the transition from step (b) to step (c)        occurs without opening the system;    -   (d) performing a second expansion by supplementing the cell        culture medium of the second population of TILs with additional        IL-2, optionally OKT-3, and antigen presenting cells (APCs), to        produce a third population of TILs, wherein the second expansion        is performed for about 7-14 days to obtain the third population        of TILs, wherein the third population of TILs is a therapeutic        population of TILs, wherein the second expansion is performed in        a closed container providing a second gas-permeable surface        area, and wherein the transition from step (c) to step (d)        occurs without opening the system;    -   (e) harvesting the therapeutic population of TILs obtained from        step (d) to provide a harvested TIL population, wherein the        transition from step (d) to step (e) occurs without opening the        system;    -   (f) transferring the harvested TIL population from step (e) to        an infusion bag, wherein the transfer from step (e) to (f)        occurs without opening the system, and optionally cryopreserving        the harvested TIL population;    -   (g) administering a therapeutically effective amount of the        harvested TIL population to the patient with double-refractory        metastatic melanoma; and    -   (h) measuring the level of IP-10 in the patient after        administering a therapeutically effective amount of the TILs in        step (g).

In an embodiment and in accordance with any of the above, an increase inthe level of IP-10 in step (h) is measured.

In an embodiment and in accordance with any of the above, the increasein the level of IP-10 is measured by calculating the difference in IP-10level in plasma seven days before TIL infusion and one day after TILinfusion, and wherein said difference in IP-10 level in plasma is atleast 800 pg/mL, at least 900 pg/mL, at least 1000 pg/mL, at least 1100pg/mL, at least 1200 pg/mL, at least 1300 pg/mL, at least 1400 pg/mL, atleast 1500 pg/mL, at least 1600 pg/mL, at least 1650 pg/mL, at least1656 pg/mL, at least 1700 pg/mL, or at least 1800 pg/mL, at least 1900pg/mL, at least 2000 pg/mL, at least 2100 pg/mL, or at least 2200 pg/mL.

In an embodiment and in accordance with any of the above, an increase inthe level of IP-10 in step (h) is indicative of treatment efficacy.

In an embodiment and in accordance with any of the above, the level ofIP-10 is measured about 1 day to 10 days post administering thetherapeutically effective amount of the TILs in step (g).

In an embodiment and in accordance with any of the above, the level ofIP-10 is measured 1 day post administering a therapeutically effectiveamount of the TILs in step (g).

In an embodiment and in accordance with any of the above, the level ofIP-10 is measured about 6 hours to 24 hours post administering thetherapeutically effective amount of the TILs in step (g).

In an embodiment and in accordance with any of the above, the methodfurther comprises a step of measuring the level of IP-10 in the patientprior to administering a therapeutically effective amount of the TILs instep (g).

In an embodiment and in accordance with any of the above, the increaseis based on an increase in the level of IP-10 after administering atherapeutically effective amount of the TILs in step (g) as compared tothe level of IP-10 in the patient prior to administering atherapeutically effective amount of the TILs in step (g).

In an embodiment and in accordance with any of the above, the methodfurther comprises step (i) predicting the patient will respond to thetherapeutically effective amount of the TILs administered in step (g)based upon measuring an increase in the level of IP-10 in step (h).

In an embodiment and in accordance with any of the above, the patient isadministered one or more further dosages of a therapeutically effectivepopulation of tumor infiltrating lymphocytes (TILs).

In an embodiment and in accordance with any of the above, the methodfurther comprises step (i) predicting the patient will not respond tothe therapeutically effective amount of the TILs administered in step(g) based upon measuring no increase in the level of IP-10 in step (h).

In an embodiment and in accordance with any of the above, the methodfurther comprises step (i) predicting the patient will respond to thetherapeutically effective amount of the TILs administered in step (g)based upon measuring an increase in the level of IP-10 in step (h) orpredicting the patient will not respond to the therapeutically effectiveamount of the TILs administered in step (g) based upon measuring noincrease in the level of IP-10 in step (h).

In an embodiment and in accordance with any of the above, predicting theprobability that the patient will or will not respond to thetherapeutically effective amount of the TILs administered in step (g) isbased upon the presence or absence of an increase in the level of IP-10in step (h).

In an embodiment and in accordance with any of the above, the increasein the level of IP-10 is an increase of at least one-fold, two-fold,three-fold, four-fold, or five-fold or more.

In an embodiment and in accordance with any of the above, predicting theprobability that the patient will or will not respond to thetherapeutically effective amount of the TILs administered in step (g)comprises correlating the level of IP-10 measured in the patient with athreshold value, wherein if the level of IP-10 measured is above thethreshold value one or more further TIL treatment dosages is indicated.

In some embodiments, the invention provides a method of predicting atreatment response and/or predicting treatment efficacy foradministration of a therapeutically effective amount of tumorinfiltrating lymphocytes (TILs) to a patient, the method comprising:

-   -   a) obtaining a biological sample from a patient with cancer,        including double-refractory metastatic melanoma;    -   b) measuring the level of IP-10 in the biological sample from        a);    -   c) administering a therapeutically effective amount of TILs;    -   d) obtaining a biological sample from the patient after the        administration of the therapeutically effective amount of TILs        in step c)    -   e) measuring the level of IP-10 in the biological sample from        d);    -   f) predicting a treatment response to and/or predicting        treatment efficacy of the administration of the therapeutically        effective amount of the TILs based upon the level of IP-10        measured after administration as compared to the level of IP-10        measured prior to administration.

In an embodiment and in accordance with any of the above, an increase inthe level of IP-10 measured in step (e) as compared to the level ofIP-10 measured step (b) is observed.

In an embodiment and in accordance with any of the above, the increasein the level of IP-10 is measured by calculating the difference in IP-10level in plasma seven days before TIL infusion and one day after TILinfusion, and wherein said difference in IP-10 level in plasma is atleast 800 pg/mL, at least 900 pg/mL, at least 1000 pg/mL, at least 1100pg/mL, at least 1200 pg/mL, at least 1300 pg/mL, at least 1400 pg/mL, atleast 1500 pg/mL, at least 1600 pg/mL, at least 1650 pg/mL, at least1656 pg/mL, at least 1700 pg/mL, or at least 1800 pg/mL, at least 1900pg/mL, at least 2000 pg/mL, at least 2100 pg/mL, or at least 2200 pg/mL.

In an embodiment and in accordance with any of the above, an increase inthe level of IP-10 in step (e) as compared to the level of IP-10measured step (b) is indicative of treatment efficacy.

In an embodiment and in accordance with any of the above, the level ofIP-10 is measured in step (e) about 1 day to 10 days post administeringa therapeutically effective amount of the TILs in step (c).

In an embodiment and in accordance with any of the above, the level ofIP-10 is measured in step (e) about 1 day post administering atherapeutically effective amount of the TILs in step (c).

In an embodiment and in accordance with any of the above, the level ofIP-10 is measured in step (e) about 6 hours to 24 hours postadministering a therapeutically effective amount of the TILs in step(g).

In an embodiment and in accordance with any of the above, predictingthat the patient will or will not respond to the therapeuticallyeffective amount of the TILs administered in step (c) is based upon anincrease in the level of IP-10 measured in step (f).

In an embodiment and in accordance with any of the above, measuring anincrease in the level of IP-10 measured in step (e) as compared to thelevel of IP-10 measured step (b) indicates that the patient will respondto the therapeutically effective amount of the TILs administered in step(d).

In an embodiment and in accordance with any of the above, the patient isadministered one or more further dosages of a therapeutically effectivepopulation of tumor infiltrating lymphocytes (TILs).

In an embodiment and in accordance with any of the above, measuring noincrease in the level of IP-10 measured in step (e) as compared to thelevel of IP-10 measured step (b) indicates that the patient will notrespond to the therapeutically effective amount of the TILs administeredin step (d).

In an embodiment and in accordance with any of the above, the level ofIP-10 is increased one-fold, two-fold, three-fold, four-fold, five-foldor more.

In an embodiment and in accordance with any of the above, predictingthat the patient will or will not respond to the therapeuticallyeffective amount of the TILs administered in step (d) further comprisescorrelating the level of IP-10 measured in the patient with a thresholdvalue, wherein if the level of IP-10 measured is above the thresholdvalue one or more further TIL treatment dosages is indicated.

In an embodiment and in accordance with any of the above, thetherapeutically effective population of TILs comprises from about2.3×10¹⁰ to about 13.7×10¹⁰ TILs.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing summary, as well as the following detailed description ofthe invention, will be better understood when read in conjunction withthe appended drawings.

FIG. 1 illustrates a TIL expansion and therapeutic treatment process.Step 1 refers to the addition of 4 tumor fragments into 10 G-Rex 10flasks. At step 2, approximately 40×10⁶ TILs or greater are obtained. Atstep 3, a split occurs into 36 G-Rex 100 flasks for REP. TILs areharvested by centrifugation at step 4. Fresh TIL product is obtained atstep 5 after a total process time of approximately 43 days, at whichpoint TILs may be infused into a patient.

FIG. 2 illustrates a treatment and manufacturing timeline for use withTILs prepared according to the present disclosure and the process ofFIG. 1. Surgery (and tumor resection) occurs at the start, andlymphodepletion chemo refers to non-myeloablative lymphodepletion withchemotherapy as described elsewhere herein.

FIG. 3 illustrates a TIL expansion and therapeutic treatment process,including a “direct to REP” step wherein pre-REP TILs are placeddirectly into a REP process. The total process time is approximately 22days, at which point TILs may be infused into a patient.

FIG. 4 illustrates a treatment and manufacturing timeline for use withTILs prepared according to the present disclosure and the process ofFIG. 3, when the cell count at day 6 is greater than 250×10⁶.

FIG. 5 illustrates a treatment and manufacturing timeline for use withTILs prepared according to the present disclosure and the process ofFIG. 3, when the cell count at day 6 is less than 250×10⁶, and whereinlymphodepletion is begun later so as to allow for an assessment of theviability of the TIL product before lymphodepleting the patient.

FIG. 6 shows a detailed schematic of a TIL manufacturing processaccording to FIG. 3.

FIG. 7 depicts the design of a clinical study using TIL therapiesprepared by different methods in double-refractory melanoma.

FIG. 8 summarizes patient characteristics in the clinical study.

FIG. 9 summarizes patient characteristics in the clinical study.

FIG. 10 summarizes treatment emergent serious adverse events in theclinical study. The “*” indicates a not related to therapy eventoccurring six months after treatment.

FIG. 11 summarizes efficacy results in the clinical study.

FIG. 12 illustrates a waterfall of response plot showing efficacy in theclinical study. Responses are independent of BRAF mutational status.

FIG. 13 illustrates time to best response and duration in the clinicalstudy.

FIG. 14 illustrates percentage change in sum of diameters in theclinical study.

FIG. 15 illustrates scans from a patient in complete remission.

FIG. 16 illustrates the design of a clinical study using TIL therapies.

FIG. 17 illustrates the results of a second clinical study performingusing a TIL manufacturing process as described in Radvanyi, et al.,Clin. Cancer Res. 2012, 18, 6758-70. NT refers to not tested, WT refersto wild type, and irRC refers to immune-related response criteria. Siteof TIL harvest is specified as follows: 1: skin/SC; 2: lymph nodes; 3:lungs; and 4: gastrointestinal/visceral.

FIG. 18: Exemplary Process 2A chart providing an overview of Steps Athrough F.

FIG. 19A-19C: Process Flow Chart of Process 2A.

FIG. 20: Shows a diagram of an embodiment of a cryopreserved TILexemplary manufacturing process (˜22 days).

FIG. 21: Shows a diagram of an embodiment of process 2A, a 22-dayprocess for TIL manufacturing.

FIG. 22: Comparison table of Steps A through F from exemplaryembodiments of process 1C and process 2A.

FIG. 23: Detailed comparison of an embodiment of process 1C and anembodiment of process 2A.

FIG. 24A-24B: Updated efficacy data for Cohort 1 from the final data cut(N=23 patients). Abbreviations: PR, partial response; SD, stabledisease; PD, progressive disease.

FIG. 25: Scheme of Gen 2 cryopreserved LN-144 manufacturing process.

FIG. 26: Scheme of study design of multicenter phase 2 clinical trial ofnovel cryopreserved TILs administered to patients with metastaticmelanoma.

FIG. 27: Table illustrating the Comparison Patient Characteristics fromCohort 1 (ASCO 2017) vs Cohort 2.

FIG. 28: Table illustrating treatment emergent adverse events (≥30%).

FIG. 29: Efficacy of the infusion product and TIL therapy.

FIG. 30: Clinical status of response evaluable patients with SD or abetter response.

FIG. 31: Percent change in sum of diameters.

FIG. 32: An increase of HMGB1 level was observed upon TIL treatment.

FIG. 33: An increase in the biomarker IL-10 was observed post-LN-144infusion.

FIG. 34: Updated patient characteristics for Cohort 2 of the phase 2clinical trial in metastatic melanoma from the second data cut (N=17patients).

FIG. 35: Treatment emergent adverse events for Cohort 2 (≥30%) from thesecond data cut (N=17 patients).

FIG. 36: Time to response for evaluable patients (stable disease orbetter) in Cohort 2 from the second data cut (N=17 patients). Of the 10patients in the efficacy set, one patient (Patient 10) was not evaluabledue to a melanoma-related death prior to the first tumor assessment notrepresented on the figure.

FIG. 37: Updated efficacy data for Cohort 2 from the second data cut(N=17 patients). The mean number of TILs infused is 34×10⁹. The mediannumber of prior therapies was 4.5. Patients with a BRAF mutationresponded as well as patients with wild-type BRAF (a*refers to patientswith a BRAF mutation). One patient (Patient 10) was not evaluable due toa melanoma-related death prior to the first tumor assessment but wasstill considered in the efficacy set. Abbreviations: PR, partialresponse; SD, stable disease; PD, progressive disease.

FIG. 38: Updated efficacy data for evaluable patients from Cohort 2 fromthe second data cut (N=17 patients). The * indicates a non-evaluablepatient that did not reach the first assessment. All efficacy-evaluablepatients had received prior anti-PD-1 and anti-CTLA-4 checkpointinhibitor therapies.

FIG. 39: Representative computed tomography scan of a patient (003-015)with a PR from Cohort 2, second data cut.

FIG. 40: Exemplary schematic of the process for manufacturing ofcryopreserved autologous TIL (LN-144, lifileucel), 22-day process.

FIG. 41: Schematic of the study design for example 14.

FIG. 42: Charts showing patient characteristics for Cohort 2. 3.3 meanprior therapies, ranging from 1-9. High tumor burden at baseline 112 mmsum of diameters for the target lesions.

FIG. 43: Data showing efficacy of treatment response for Example 14study. Four patients who had no disease assessment following autologousTIL (lifileucel, LN-144) due to cancer-related death are not shown. PerRECIST 1.1, two patients (31, 33) had BOR of SD: met PR criteria at Day42 and PD at Day 84 due to new lesions

FIG. 44: Data showing time to response for evaluable patients (PR orBetter). (1) BOR is best overall response on prior anti-PD-1immunotherapy. (2) U: unknown best overall response on prior anti-PD-1immunotherapy.

FIG. 45: Data showing ercent change from baseline in sum of targetlesion diameters over time.

FIG. 46: Data showing treatment emergent adverse events (≥30%). *Onedeath was due to intra-abdominal hemorrhage considered possibly relatedto TIL and one was due to acute respiratory failure assessed as notrelated to TIL per investigator assessment. Patients with multipleevents for a given preferred term are counted only once using themaximum grade under each preferred term. Treatment-Emergent AdverseEvents refer to all AEs starting on or after the first dose date of TILup to 30 days.

FIG. 47: Chart showing treatment efficacy from the Example 14 study. *NEdue to not reaching first assessment. 1 uPRs (4) were all due to timingnot having reached the second assessment.

FIG. 48: Data showing biomarker levels for IP-10. Change in IP-10(CXCL10) level in periphery may have a correlation with response. Meanchange in IP-10 levels from baseline to day 1 post TIL infusion washigher among responders vs. nonresponders (p=0.19). the Y-axis is inpg/mL. D-7 is seven days before TIL infusion (administration) and D-1 isone day after TIL infusion (administration).

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO:1 is the amino acid sequence of the heavy chain of muromonab.

SEQ ID NO:2 is the amino acid sequence of the light chain of muromonab.

SEQ ID NO:3 is the amino acid sequence of a recombinant human IL-2protein.

SEQ ID NO:4 is the amino acid sequence of aldesleukin.

SEQ ID NO:5 is the amino acid sequence of a recombinant human IL-4protein.

SEQ ID NO:6 is the amino acid sequence of a recombinant human IL-7protein.

SEQ ID NO:7 is the amino acid sequence of a recombinant human IL-15protein.

SEQ ID NO:8 is the amino acid sequence of a recombinant human IL-21protein.

SEQ ID NO:9 is the amino acid sequence of human 4-1BB.

SEQ ID NO:10 is the amino acid sequence of murine 4-1BB.

SEQ ID NO:11 is the heavy chain for the 4-1BB agonist monoclonalantibody utomilumab (PF-05082566).

SEQ ID NO:12 is the light chain for the 4-1BB agonist monoclonalantibody utomilumab (PF-05082566).

SEQ ID NO:13 is the heavy chain variable region (VH) for the 4-1BBagonist monoclonal antibody utomilumab (PF-05082566).

SEQ ID NO:14 is the light chain variable region (VL) for the 4-1BBagonist monoclonal antibody utomilumab (PF-05082566).

SEQ ID NO:15 is the heavy chain CDR1 for the 4-1BB agonist monoclonalantibody utomilumab (PF-05082566).

SEQ ID NO:16 is the heavy chain CDR2 for the 4-1BB agonist monoclonalantibody utomilumab (PF-05082566).

SEQ ID NO:17 is the heavy chain CDR3 for the 4-1BB agonist monoclonalantibody utomilumab (PF-05082566).

SEQ ID NO:18 is the light chain CDR1 for the 4-1BB agonist monoclonalantibody utomilumab (PF-05082566).

SEQ ID NO:19 is the light chain CDR2 for the 4-1BB agonist monoclonalantibody utomilumab (PF-05082566).

SEQ ID NO:20 is the light chain CDR3 for the 4-1BB agonist monoclonalantibody utomilumab (PF-05082566).

SEQ ID NO:21 is the heavy chain for the 4-1BB agonist monoclonalantibody urelumab (BMS-663513).

SEQ ID NO:22 is the light chain for the 4-1BB agonist monoclonalantibody urelumab (BMS-663513).

SEQ ID NO:23 is the heavy chain variable region (VH) for the 4-1BBagonist monoclonal antibody urelumab (BMS-663513).

SEQ ID NO:24 is the light chain variable region (VL) for the 4-1BBagonist monoclonal antibody urelumab (BMS-663513).

SEQ ID NO:25 is the heavy chain CDR1 for the 4-1BB agonist monoclonalantibody urelumab (BMS-663513).

SEQ ID NO:26 is the heavy chain CDR2 for the 4-1BB agonist monoclonalantibody urelumab (BMS-663513).

SEQ ID NO:27 is the heavy chain CDR3 for the 4-1BB agonist monoclonalantibody urelumab (BMS-663513).

SEQ ID NO:28 is the light chain CDR1 for the 4-1BB agonist monoclonalantibody urelumab (BMS-663513).

SEQ ID NO:29 is the light chain CDR2 for the 4-1BB agonist monoclonalantibody urelumab (BMS-663513).

SEQ ID NO:30 is the light chain CDR3 for the 4-1BB agonist monoclonalantibody urelumab (BMS-663513).

SEQ ID NO:31 is an Fc domain for a TNFRSF agonist fusion protein.

SEQ ID NO:32 is a linker for a TNFRSF agonist fusion protein.

SEQ ID NO:33 is a linker for a TNFRSF agonist fusion protein.

SEQ ID NO:34 is a linker for a TNFRSF agonist fusion protein.

SEQ ID NO:35 is a linker for a TNFRSF agonist fusion protein.

SEQ ID NO:36 is a linker for a TNFRSF agonist fusion protein.

SEQ ID NO:37 is a linker for a TNFRSF agonist fusion protein.

SEQ ID NO:38 is a linker for a TNFRSF agonist fusion protein.

SEQ ID NO:39 is a linker for a TNFRSF agonist fusion protein.

SEQ ID NO:40 is a linker for a TNFRSF agonist fusion protein.

SEQ ID NO:41 is a linker for a TNFRSF agonist fusion protein.

SEQ ID NO:42 is an Fc domain for a TNFRSF agonist fusion protein.

SEQ ID NO:43 is a linker for a TNFRSF agonist fusion protein.

SEQ ID NO:44 is a linker for a TNFRSF agonist fusion protein.

SEQ ID NO:45 is a linker for a TNFRSF agonist fusion protein.

SEQ ID NO:46 is a 4-1BB ligand (4-1BBL) amino acid sequence.

SEQ ID NO:47 is a soluble portion of 4-1BBL polypeptide.

SEQ ID NO:48 is a heavy chain variable region (VH) for the 4-1BB agonistantibody 4B4-1-1 version 1.

SEQ ID NO:49 is a light chain variable region (VL) for the 4-1BB agonistantibody 4B4-1-1 version 1.

SEQ ID NO:50 is a heavy chain variable region (VH) for the 4-1BB agonistantibody 4B4-1-1 version 2.

SEQ ID NO:51 is a light chain variable region (VL) for the 4-1BB agonistantibody 4B4-1-1 version 2.

SEQ ID NO:52 is a heavy chain variable region (VH) for the 4-1BB agonistantibody H39E3-2.

SEQ ID NO:53 is a light chain variable region (VL) for the 4-1BB agonistantibody H39E3-2.

SEQ ID NO:54 is the amino acid sequence of human OX40.

SEQ ID NO:55 is the amino acid sequence of murine OX40.

SEQ ID NO:56 is the heavy chain for the OX40 agonist monoclonal antibodytavolixizumab (MEDI-0562).

SEQ ID NO:57 is the light chain for the OX40 agonist monoclonal antibodytavolixizumab (MEDI-0562).

SEQ ID NO:58 is the heavy chain variable region (VH) for the OX40agonist monoclonal antibody tavolixizumab (MEDI-0562).

SEQ ID NO:59 is the light chain variable region (VL) for the OX40agonist monoclonal antibody tavolixizumab (MEDI-0562).

SEQ ID NO:60 is the heavy chain CDR1 for the OX40 agonist monoclonalantibody tavolixizumab (MEDI-0562).

SEQ ID NO:61 is the heavy chain CDR2 for the OX40 agonist monoclonalantibody tavolixizumab (MEDI-0562).

SEQ ID NO:62 is the heavy chain CDR3 for the OX40 agonist monoclonalantibody tavolixizumab (MEDI-0562).

SEQ ID NO:63 is the light chain CDR1 for the OX40 agonist monoclonalantibody tavolixizumab (MEDI-0562).

SEQ ID NO:64 is the light chain CDR2 for the OX40 agonist monoclonalantibody tavolixizumab (MEDI-0562).

SEQ ID NO:65 is the light chain CDR3 for the OX40 agonist monoclonalantibody tavolixizumab (MEDI-0562).

SEQ ID NO:66 is the heavy chain for the OX40 agonist monoclonal antibody11D4.

SEQ ID NO:67 is the light chain for the OX40 agonist monoclonal antibody11D4.

SEQ ID NO:68 is the heavy chain variable region (VH) for the OX40agonist monoclonal antibody 11D4.

SEQ ID NO:69 is the light chain variable region (VL) for the OX40agonist monoclonal antibody 11D4.

SEQ ID NO:70 is the heavy chain CDR1 for the OX40 agonist monoclonalantibody 11D4.

SEQ ID NO:71 is the heavy chain CDR2 for the OX40 agonist monoclonalantibody 11D4.

SEQ ID NO:72 is the heavy chain CDR3 for the OX40 agonist monoclonalantibody 11D4.

SEQ ID NO:73 is the light chain CDR1 for the OX40 agonist monoclonalantibody 11D4.

SEQ ID NO:74 is the light chain CDR2 for the OX40 agonist monoclonalantibody 11D4.

SEQ ID NO:75 is the light chain CDR3 for the OX40 agonist monoclonalantibody 11D4.

SEQ ID NO:76 is the heavy chain for the OX40 agonist monoclonal antibody18D8.

SEQ ID NO:77 is the light chain for the OX40 agonist monoclonal antibody18D8.

SEQ ID NO:78 is the heavy chain variable region (VH) for the OX40agonist monoclonal antibody 18D8.

SEQ ID NO:79 is the light chain variable region (VL) for the OX40agonist monoclonal antibody 18D8.

SEQ ID NO:80 is the heavy chain CDR1 for the OX40 agonist monoclonalantibody 18D8.

SEQ ID NO:81 is the heavy chain CDR2 for the OX40 agonist monoclonalantibody 18D8.

SEQ ID NO:82 is the heavy chain CDR3 for the OX40 agonist monoclonalantibody 18D8.

SEQ ID NO:83 is the light chain CDR1 for the OX40 agonist monoclonalantibody 18D8.

SEQ ID NO:84 is the light chain CDR2 for the OX40 agonist monoclonalantibody 18D8.

SEQ ID NO:85 is the light chain CDR3 for the OX40 agonist monoclonalantibody 18D8.

SEQ ID NO:86 is the heavy chain variable region (VH) for the OX40agonist monoclonal antibody Hu119-122.

SEQ ID NO:87 is the light chain variable region (VL) for the OX40agonist monoclonal antibody Hu119-122.

SEQ ID NO:88 is the heavy chain CDR1 for the OX40 agonist monoclonalantibody Hu119-122.

SEQ ID NO:89 is the heavy chain CDR2 for the OX40 agonist monoclonalantibody Hu119-122.

SEQ ID NO:90 is the heavy chain CDR3 for the OX40 agonist monoclonalantibody Hu119-122.

SEQ ID NO:91 is the light chain CDR1 for the OX40 agonist monoclonalantibody Hu119-122.

SEQ ID NO:92 is the light chain CDR2 for the OX40 agonist monoclonalantibody Hu119-122.

SEQ ID NO:93 is the light chain CDR3 for the OX40 agonist monoclonalantibody Hu119-122.

SEQ ID NO:94 is the heavy chain variable region (VH) for the OX40agonist monoclonal antibody Hu106-222.

SEQ ID NO:95 is the light chain variable region (VL) for the OX40agonist monoclonal antibody Hu106-222.

SEQ ID NO:96 is the heavy chain CDR1 for the OX40 agonist monoclonalantibody Hu106-222.

SEQ ID NO:97 is the heavy chain CDR2 for the OX40 agonist monoclonalantibody Hu106-222.

SEQ ID NO:98 is the heavy chain CDR3 for the OX40 agonist monoclonalantibody Hu106-222.

SEQ ID NO:99 is the light chain CDR1 for the OX40 agonist monoclonalantibody Hu106-222.

SEQ ID NO:100 is the light chain CDR2 for the OX40 agonist monoclonalantibody Hu106-222.

SEQ ID NO:101 is the light chain CDR3 for the OX40 agonist monoclonalantibody Hu106-222.

SEQ ID NO:102 is an OX40 ligand (OX40L) amino acid sequence.

SEQ ID NO:103 is a soluble portion of OX40L polypeptide.

SEQ ID NO:104 is an alternative soluble portion of OX40L polypeptide.

SEQ ID NO:105 is the heavy chain variable region (VH) for the OX40agonist monoclonal antibody 008.

SEQ ID NO:106 is the light chain variable region (VL) for the OX40agonist monoclonal antibody 008.

SEQ ID NO:107 is the heavy chain variable region (VH) for the OX40agonist monoclonal antibody 011.

SEQ ID NO:108 is the light chain variable region (VL) for the OX40agonist monoclonal antibody 011.

SEQ ID NO:109 is the heavy chain variable region (VH) for the OX40agonist monoclonal antibody 021.

SEQ ID NO:110 is the light chain variable region (VL) for the OX40agonist monoclonal antibody 021.

SEQ ID NO:111 is the heavy chain variable region (VH) for the OX40agonist monoclonal antibody 023.

SEQ ID NO:112 is the light chain variable region (VL) for the OX40agonist monoclonal antibody 023.

SEQ ID NO:113 is the heavy chain variable region (VH) for an OX40agonist monoclonal antibody.

SEQ ID NO:114 is the light chain variable region (VL) for an OX40agonist monoclonal antibody.

SEQ ID NO:115 is the heavy chain variable region (VH) for an OX40agonist monoclonal antibody.

SEQ ID NO:116 is the light chain variable region (VL) for an OX40agonist monoclonal antibody.

SEQ ID NO:117 is the heavy chain variable region (VH) for a humanizedOX40 agonist monoclonal antibody.

SEQ ID NO:118 is the heavy chain variable region (VH) for a humanizedOX40 agonist monoclonal antibody.

SEQ ID NO:119 is the light chain variable region (VL) for a humanizedOX40 agonist monoclonal antibody.

SEQ ID NO:120 is the light chain variable region (VL) for a humanizedOX40 agonist monoclonal antibody.

SEQ ID NO:121 is the heavy chain variable region (VH) for a humanizedOX40 agonist monoclonal antibody.

SEQ ID NO:122 is the heavy chain variable region (VH) for a humanizedOX40 agonist monoclonal antibody.

SEQ ID NO:123 is the light chain variable region (VL) for a humanizedOX40 agonist monoclonal antibody.

SEQ ID NO:124 is the light chain variable region (VL) for a humanizedOX40 agonist monoclonal antibody.

SEQ ID NO:125 is the heavy chain variable region (VH) for an OX40agonist monoclonal antibody.

SEQ ID NO:126 is the light chain variable region (VL) for an OX40agonist monoclonal antibody.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which this invention belongs. All patents and publicationsreferred to herein are incorporated by reference in their entireties.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which this invention belongs. All patents and publicationsreferred to herein are incorporated by reference in their entireties.

The term “in vivo” refers to an event that takes place in a subject'sbody.

The term “in vitro” refers to an event that takes places outside of asubject's body. In vitro assays encompass cell-based assays in whichcells alive or dead are employed and may also encompass a cell-freeassay in which no intact cells are employed.

The term “ex vivo” refers to an event which involves treating orperforming a procedure on a cell, tissue and/or organ which has beenremoved from a subject's body. Aptly, the cell, tissue and/or organ maybe returned to the subject's body in a method of surgery or treatment.

The term “rapid expansion” means an increase in the number ofantigen-specific TILs of at least about 3-fold (or 4-, 5-, 6-, 7-, 8-,or 9-fold) over a period of a week, more preferably at least about10-fold (or 20-, 30-, 40-, 50-, 60-, 70-, 80-, or 90-fold) over a periodof a week, or most preferably at least about 100-fold over a period of aweek. A number of rapid expansion protocols are outlined below.

By “tumor infiltrating lymphocytes” or “TILs” herein is meant apopulation of cells originally obtained as white blood cells that haveleft the bloodstream of a subject and migrated into a tumor. TILsinclude, but are not limited to, CD8+ cytotoxic T cells (lymphocytes),Th1 and Th17 CD4+ T cells, natural killer cells, dendritic cells and M1macrophages. TILs include both primary and secondary TILs. “PrimaryTILs” are those that are obtained from patient tissue samples asoutlined herein (sometimes referred to as “freshly harvested”), and“secondary TILs” are any TIL cell populations that have been expanded orproliferated as discussed herein, including, but not limited to bulkTILs and expanded TILs (“REP TILs” or “post-REP TILs”). TIL cellpopulations can include genetically modified TILs.

By “population of cells” (including TILs) herein is meant a number ofcells that share common traits. In general, populations generally rangefrom 1×10⁶ to 1×10¹⁰ in number, with different TIL populationscomprising different numbers. For example, initial growth of primaryTILs in the presence of IL-2 results in a population of bulk TILs ofroughly 1×10⁸ cells. REP expansion is generally done to providepopulations of 1.5×10⁹ to 1.5×10¹⁰ cells for infusion.

By “cryopreserved TILs” herein is meant that TILs, either primary, bulk,or expanded (REP TILs), are treated and stored in the range of about−150° C. to −60° C. General methods for cryopreservation are alsodescribed elsewhere herein, including in the Examples. For clarity,“cryopreserved TILs” are distinguishable from frozen tissue sampleswhich may be used as a source of primary TILs.

By “thawed cryopreserved TILs” herein is meant a population of TILs thatwas previously cryopreserved and then treated to return to roomtemperature or higher, including but not limited to cell culturetemperatures or temperatures wherein TILs may be administered to apatient.

TILs can generally be defined either biochemically, using cell surfacemarkers, or functionally, by their ability to infiltrate tumors andeffect treatment. TILs can be generally categorized by expressing one ormore of the following biomarkers: CD4, CD8, TCR αβ, CD27, CD28, CD56,CCR7, CD45Ra, CD95, PD-1, and CD25. Additionally and alternatively, TILscan be functionally defined by their ability to infiltrate solid tumorsupon reintroduction into a patient.

The term “cryopreservation media” or “cryopreservation medium” refers toany medium that can be used for cryopreservation of cells. Such mediacan include media comprising 7% to 10% DMSO. Exemplary media includeCryoStor CS10, Hyperthermasol, as well as combinations thereof. The term“CS10” refers to a cryopreservation medium which is obtained fromStemcell Technologies or from Biolife Solutions. The CS10 medium may bereferred to by the trade name “CryoStor® CS10”. The CS10 medium is aserum-free, animal component-free medium which comprises DMSO.

The term “central memory T cell” refers to a subset of T cells that inthe human are CD45R0+ and constitutively express CCR7 (CCR7hi) and CD62L(CD62hi). The surface phenotype of central memory T cells also includesTCR, CD3, CD127 (IL-7R), and IL-15R. Transcription factors for centralmemory T cells include BCL-6, BCL-6B, MBD2, and BMI1. Central memory Tcells primarily secret IL-2 and CD40L as effector molecules after TCRtriggering. Central memory T cells are predominant in the CD4compartment in blood, and in the human are proportionally enriched inlymph nodes and tonsils.

The term “effector memory T cell” refers to a subset of human ormammalian T cells that, like central memory T cells, are CD45R0+, buthave lost the constitutive expression of CCR7 (CCR7lo) and areheterogeneous or low for CD62L expression (CD62Llo). The surfacephenotype of central memory T cells also includes TCR, CD3, CD127(IL-7R), and IL-15R. Transcription factors for central memory T cellsinclude BLIMP1. Effector memory T cells rapidly secret high levels ofinflammatory cytokines following antigenic stimulation, includinginterferon-γ, IL-4, and IL-5. Effector memory T cells are predominant inthe CD8 compartment in blood, and in the human are proportionallyenriched in the lung, liver, and gut. CD8+ effector memory T cells carrylarge amounts of perforin.

The term “closed system” refers to a system that is closed to theoutside environment. Any closed system appropriate for cell culturemethods can be employed with the methods of the present invention.Closed systems include, for example, but are not limited to closedG-containers. Once a tumor segment is added to the closed system, thesystem is no opened to the outside environment until the TILs are readyto be administered to the patient.

The terms “fragmenting,” “fragment,” and “fragmented,” as used herein todescribe processes for disrupting a tumor, includes mechanicalfragmentation methods such as crushing, slicing, dividing, andmorcellating tumor tissue as well as any other method for disrupting thephysical structure of tumor tissue.

The terms “peripheral blood mononuclear cells” and “PBMCs” refers to aperipheral blood cell having a round nucleus, including lymphocytes (Tcells, B cells, NK cells) and monocytes. Preferably, the peripheralblood mononuclear cells are irradiated allogeneic peripheral bloodmononuclear cells. PBMCs are a type of antigen-presenting cell.

The term “anti-CD3 antibody” refers to an antibody or variant thereof,e.g., a monoclonal antibody and including human, humanized, chimeric ormurine antibodies which are directed against the CD3 receptor in the Tcell antigen receptor of mature T cells. Anti-CD3 antibodies includeOKT-3, also known as muromonab. Anti-CD3 antibodies also include theUHCT1 clone, also known as T3 and CD3c. Other anti-CD3 antibodiesinclude, for example, otelixizumab, teplizumab, and visilizumab.

The term “OKT-3” (also referred to herein as “OKT3”) refers to amonoclonal antibody or biosimilar or variant thereof, including human,humanized, chimeric, or murine antibodies, directed against the CD3receptor in the T cell antigen receptor of mature T cells, and includescommercially-available forms such as OKT-3 (30 ng/mL, MACS GMP CD3 pure,Miltenyi Biotech, Inc., San Diego, Calif., USA) and muromonab orvariants, conservative amino acid substitutions, glycoforms, orbiosimilars thereof. The amino acid sequences of the heavy and lightchains of muromonab are given in Table 1 (SEQ ID NO:1 and SEQ ID NO:2).A hybridoma capable of producing OKT-3 is deposited with the AmericanType Culture Collection and assigned the ATCC accession number CRL 8001.A hybridoma capable of producing OKT-3 is also deposited with EuropeanCollection of Authenticated Cell Cultures (ECACC) and assigned CatalogueNo. 86022706. A hybridoma capable of producing OKT-3 is deposited withthe American Type Culture Collection and assigned the ATCC accessionnumber CRL 8001. A hybridoma capable of producing OKT-3 is alsodeposited with European Collection of Authenticated Cell Cultures(ECACC) and assigned Catalogue No. 86022706. Anti-CD3 antibodies alsoinclude the UHCT1 clone (commercially available from BioLegend, SanDiego, Calif., USA), also known as T3 and CD3ε.

TABLE 1 Amino acid sequences of muromonab. Identifier Sequence(One-Letter Amino Acid Symbols) SEQ ID NO: 1 QVQLQQSGAE LARPGASVKMSCKASGYTFT RYTMHWVKQR PGQGLEWIGY INPSRGYTNY 60 Muromonab heavyNQKFKDKATL TTDKSSSTAY MQLSSLTSED SAVYYCARYY DDHYCLDYWG QGTTLTVSSA 120chain KTTAPSVYPL APVCGGTTGS SVTLGCLVKG YFPEPVTLTW NSGSLSSGVH TFPAVLQSDL180 YTLSSSVTVT SSTWPSQSIT CNVAHPASST KVDKKIEPRP ESCDKTHTCP PCPAPELLGG240 PSVFLFPPKP EDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN300 STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KARGQPREPQ VYTLPPSRDE360 LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW420 QQGNVFSCSV MHEALHNHYT QKSLSLSPGK 450 SEQ ID NO: 2 QIVLTQSPAIMSASPGEKVT MTCSASSSVS YMNWYQQKSG TSPKRWIYDT SKLASGVPAH 60 Muromonablight FRGSGSGTSY SLTISGMEAE DAATYYCQQW SSNPFTFGSG TKLEINRADT APTVSIFPPS120 chain SEQLTSGGAS VVCFLNNFYP KDINVKWKID GSERQNGVLN SWTDQDSKDSTYSMSSTLTL 180 TKDEYERHNS YTCEATHETS TSPIVKSFNR NEC 213

The term “IL-2” (also referred to herein as “IL2”) refers to the T cellgrowth factor known as interleukin-2, and includes all forms of IL-2including human and mammalian forms, conservative amino acidsubstitutions, glycoforms, biosimilars, and variants thereof. IL-2 isdescribed, e.g., in Nelson, J. Immunol. 2004, 172, 3983-88 and Malek,Annu. Rev. Immunol. 2008, 26, 453-79, the disclosures of which areincorporated by reference herein. The amino acid sequence of recombinanthuman IL-2 suitable for use in the invention is given in Table 2 (SEQ IDNO:3). For example, the term IL-2 encompasses human, recombinant formsof IL-2 such as aldesleukin (PROLEUKIN, available commercially frommultiple suppliers in 22 million IU per single use vials), as well asthe form of recombinant IL-2 commercially supplied by CellGenix, Inc.,Portsmouth, N.H., USA (CELLGRO GMP) or ProSpec-Tany TechnoGene Ltd.,East Brunswick, N.J., USA (Cat. No. CYT-209-b) and other commercialequivalents from other vendors. Aldesleukin (des-alanyl-1, serine-125human IL-2) is a nonglycosylated human recombinant form of IL-2 with amolecular weight of approximately 15 kDa. The amino acid sequence ofaldesleukin suitable for use in the invention is given in Table 2 (SEQID NO:4). The term IL-2 also encompasses pegylated forms of IL-2, asdescribed herein, including the pegylated IL2 prodrug NKTR-214,available from Nektar Therapeutics, South San Francisco, Calif., USA.NKTR-214 and pegylated IL-2 suitable for use in the invention isdescribed in U.S. Patent Application Publication No. US 2014/0328791 A1and International Patent Application Publication No. WO 2012/065086 A1,the disclosures of which are incorporated by reference herein.Alternative forms of conjugated IL-2 suitable for use in the inventionare described in U.S. Pat. Nos. 4,766,106, 5,206,344, 5,089,261 and4,902,502, the disclosures of which are incorporated by referenceherein. Formulations of IL-2 suitable for use in the invention aredescribed in U.S. Pat. No. 6,706,289, the disclosure of which isincorporated by reference herein.

TABLE 2 Amino acid sequences of interleukins. Identifier Sequence(One-Letter Amino Acid Symbols) SEQ ID NO: 3 MAPTSSSTKK TQLQLEHLLLDLQMILNGIN NYENPKLTRM LTFKFYMPKK ATELKHLQCL 60 recombinant EEELKPLEEVLNLAQSKNFH LRPRDLISNI NVIVLELKGS ETTFMCEYAD ETATIVEFLN 120 human IL-2RWITFCQSII STLT 134 (rhIL-2) SEQ ID NO: 4 PTSSSTKKTQ LQLEHLLLDLQMILNGINNY KNPKLTRMLT FKEYMPKKAT ELKHLQCLEE 60 Aldesleukin ELKPLEEVLNLAQSKNFHLR PRDLISNINV IVLELKGSET TFMCEYADET ATIVEFLNRW 120 ITFSQSIIST LT132 SEQ ID NO: 5 MHKCDITLQE IIKTLNSLTE QKTLCTELTV TDIFAASENT TEKETFCRAATVLRQFYSHH 60 recombinant EKDTRCLGAT AQQFHRHKQL IRFLKRLDRN LWGLAGLNSCPVKEANQSTL ENFLERLKTI 120 human IL-4 MREKYSKCSS 130 (rhIL-4) SEQ ID NO:6 MDCDIEGKDG EQYESVLMVS IDQLLDSMKE IGSNCLNNEF NFFERHICDA NKEGMFLFRA 60recombinant ARKLRQFLKM NSTGDFDLHL LEVSEGTTIL LNCTGQVKGR KPAALGEAQPTKSLEENKSL 120 human IL-7 KEQKKLNDLC FLKRLLQEIK TCWNKILMGT KEH 153(rhIL-7) SEQ ID NO: 7 MNWVNVISDL KKIEDLIQSM HIDATLYTES DVHPSCKVTAMKCFLLELQV ISLESGDASI 60 recombinant HDTVENLIIL ANNSLSSNGN VTESGCKECEELEEKNIKEF LQSFVHIVQM FINTS 115 human IL-15 (rhIL-15) SEQ ID NO: 8MQDRHMIRMR QLIDIVDQLK NYVNDLVPEF LPAPEDVETN CEWSAFSCFQ KAQLKSANTG 60recombinant NNERIINVSI KELKRKPPST NAGRRQKHRL TCPSCDSYEK EPPKEFLERFESLLQHMIHQ 120 human IL-21 HLSSRTHGSE DS 132 (rhIL-21)

The term “IL-4” (also referred to herein as “IL4”) refers to thecytokine known as interleukin 4, which is produced by Th2 T cells and byeosinophils, basophils, and mast cells. IL-4 regulates thedifferentiation of naïve helper T cells (Th0 cells) to Th2 T cells.Steinke and Borish, Respir. Res. 2001, 2, 66-70. Upon activation byIL-4, Th2 T cells subsequently produce additional IL-4 in a positivefeedback loop. IL-4 also stimulates B cell proliferation and class IIMHC expression, and induces class switching to IgE and IgG1 expressionfrom B cells. Recombinant human IL-4 suitable for use in the inventionis commercially available from multiple suppliers, includingProSpec-Tany TechnoGene Ltd., East Brunswick, N.J., USA (Cat. No.CYT-211) and ThermoFisher Scientific, Inc., Waltham, Mass., USA (humanIL-15 recombinant protein, Cat. No. Gibco CTP0043). The amino acidsequence of recombinant human IL-4 suitable for use in the invention isgiven in Table 2 (SEQ ID NO:5).

The term “IL-7” (also referred to herein as “IL7”) refers to aglycosylated tissue-derived cytokine known as interleukin 7, which maybe obtained from stromal and epithelial cells, as well as from dendriticcells. Fry and Mackall, Blood 2002, 99, 3892-904. IL-7 can stimulate thedevelopment of T cells. IL-7 binds to the IL-7 receptor, a heterodimerconsisting of IL-7 receptor alpha and common gamma chain receptor, whichin a series of signals important for T cell development within thethymus and survival within the periphery. Recombinant human IL-7suitable for use in the invention is commercially available frommultiple suppliers, including ProSpec-Tany TechnoGene Ltd., EastBrunswick, N.J., USA (Cat. No. CYT-254) and ThermoFisher Scientific,Inc., Waltham, Mass., USA (human IL-15 recombinant protein, Cat. No.Gibco PHC0071). The amino acid sequence of recombinant human IL-7suitable for use in the invention is given in Table 2 (SEQ ID NO:6).

The term “IL-15” (also referred to herein as “IL15”) refers to the Tcell growth factor known as interleukin-15, and includes all forms ofIL-2 including human and mammalian forms, conservative amino acidsubstitutions, glycoforms, biosimilars, and variants thereof. IL-15 isdescribed, e.g., in Fehniger and Caligiuri, Blood 2001, 97, 14-32, thedisclosure of which is incorporated by reference herein. IL-15 shares βand γ signaling receptor subunits with IL-2. Recombinant human IL-15 isa single, non-glycosylated polypeptide chain containing 114 amino acids(and an N-terminal methionine) with a molecular mass of 12.8 kDa.Recombinant human IL-15 is commercially available from multiplesuppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, N.J.,USA (Cat. No. CYT-230-b) and ThermoFisher Scientific, Inc., Waltham,Mass., USA (human IL-15 recombinant protein, Cat. No. 34-8159-82). Theamino acid sequence of recombinant human IL-15 suitable for use in theinvention is given in Table 2 (SEQ ID NO:7).

The term “IL-21” (also referred to herein as “IL21”) refers to thepleiotropic cytokine protein known as interleukin-21, and includes allforms of IL-21 including human and mammalian forms, conservative aminoacid substitutions, glycoforms, biosimilars, and variants thereof. IL-21is described, e.g., in Spolski and Leonard, Nat. Rev. Drug. Disc. 2014,13, 379-95, the disclosure of which is incorporated by reference herein.IL-21 is primarily produced by natural killer T cells and activatedhuman CD4+ T cells. Recombinant human IL-21 is a single,non-glycosylated polypeptide chain containing 132 amino acids with amolecular mass of 15.4 kDa. Recombinant human IL-21 is commerciallyavailable from multiple suppliers, including ProSpec-Tany TechnoGeneLtd., East Brunswick, N.J., USA (Cat. No. CYT-408-b) and ThermoFisherScientific, Inc., Waltham, Mass., USA (human IL-21 recombinant protein,Cat. No. 14-8219-80). The amino acid sequence of recombinant human IL-21suitable for use in the invention is given in Table 2 (SEQ ID NO:8).

When “an anti-tumor effective amount”, “an tumor-inhibiting effectiveamount”, or “therapeutic amount” is indicated, the precise amount of thecompositions of the present invention to be administered can bedetermined by a physician with consideration of individual differencesin age, weight, tumor size, extent of infection or metastasis, andcondition of the patient (subject). It can generally be stated that apharmaceutical composition comprising the tumor infiltrating lymphocytes(e.g. secondary TILs or genetically modified cytotoxic lymphocytes)described herein may be administered at a dosage of 10⁴ to 10¹¹ cells/kgbody weight (e.g., 10⁵ to 10⁶, 10⁵ to 10¹⁰, 10⁵ to 10¹¹, 10⁶ to 10¹⁰,10⁶ to 10¹¹, 10⁷ to 10¹¹, 10⁷ to 10¹⁰, 10⁸ to 10¹¹, 10⁸ to 10¹⁰, 10⁹ to10¹¹, or 10⁹ to 10¹⁰ cells/kg body weight), including all integer valueswithin those ranges. Tumor infiltrating lymphocytes (including in somecases, genetically modified cytotoxic lymphocytes) compositions may alsobe administered multiple times at these dosages. The tumor infiltratinglymphocytes (including in some cases, genetically) can be administeredby using infusion techniques that are commonly known in immunotherapy(see, e.g., Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988). Theoptimal dosage and treatment regime for a particular patient can readilybe determined by one skilled in the art of medicine by monitoring thepatient for signs of disease and adjusting the treatment accordingly.

The term “hematological malignancy” refers to mammalian cancers andtumors of the hematopoietic and lymphoid tissues, including but notlimited to tissues of the blood, bone marrow, lymph nodes, and lymphaticsystem. Hematological malignancies are also referred to as “liquidtumors.” Hematological malignancies include, but are not limited to,acute lymphoblastic leukemia (ALL), chronic lymphocytic lymphoma (CLL),small lymphocytic lymphoma (SLL), acute myelogenous leukemia (AML),chronic myelogenous leukemia (CML), acute monocytic leukemia (AMoL),Hodgkin's lymphoma, and non-Hodgkin's lymphomas. The term “B cellhematological malignancy” refers to hematological malignancies thataffect B cells.

The term “solid tumor” refers to an abnormal mass of tissue that usuallydoes not contain cysts or liquid areas. Solid tumors may be benign ormalignant. The term “solid tumor cancer refers to malignant, neoplastic,or cancerous solid tumors. Solid tumor cancers include, but are notlimited to, sarcomas, carcinomas, and lymphomas, such as cancers of thelung, breast, prostate, colon, rectum, and bladder. The tissue structureof solid tumors includes interdependent tissue compartments includingthe parenchyma (cancer cells) and the supporting stromal cells in whichthe cancer cells are dispersed and which may provide a supportingmicroenvironment.

The term “liquid tumor” refers to an abnormal mass of cells that isfluid in nature. Liquid tumor cancers include, but are not limited to,leukemias, myelomas, and lymphomas, as well as other hematologicalmalignancies. TILs obtained from liquid tumors may also be referred toherein as marrow infiltrating lymphocytes (MILs).

The term “microenvironment,” as used herein, may refer to the solid orhematological tumor microenvironment as a whole or to an individualsubset of cells within the microenvironment. The tumor microenvironment,as used herein, refers to a complex mixture of “cells, soluble factors,signaling molecules, extracellular matrices, and mechanical cues thatpromote neoplastic transformation, support tumor growth and invasion,protect the tumor from host immunity, foster therapeutic resistance, andprovide niches for dominant metastases to thrive,” as described inSwartz, et al., Cancer Res., 2012, 72, 2473. Although tumors expressantigens that should be recognized by T cells, tumor clearance by theimmune system is rare because of immune suppression by themicroenvironment.

The terms “co-administration,” “co-administering,” “administered incombination with,” “administering in combination with,” “simultaneous,”and “concurrent,” as used herein, encompass administration of two ormore active pharmaceutical ingredients (in a preferred embodiment of thepresent invention, for example, at least one potassium channel agonistin combination with a plurality of TILs) to a subject so that bothactive pharmaceutical ingredients and/or their metabolites are presentin the subject at the same time. Co-administration includes simultaneousadministration in separate compositions, administration at differenttimes in separate compositions, or administration in a composition inwhich two or more active pharmaceutical ingredients are present.Simultaneous administration in separate compositions and administrationin a composition in which both agents are present are preferred.

The term “effective amount” or “therapeutically effective amount” refersto that amount of a compound or combination of compounds as describedherein that is sufficient to effect the intended application including,but not limited to, disease treatment. A therapeutically effectiveamount may vary depending upon the intended application (in vitro or invivo), or the subject and disease condition being treated (e.g., theweight, age and gender of the subject), the severity of the diseasecondition, or the manner of administration. The term also applies to adose that will induce a particular response in target cells (e.g., thereduction of platelet adhesion and/or cell migration). The specific dosewill vary depending on the particular compounds chosen, the dosingregimen to be followed, whether the compound is administered incombination with other compounds, timing of administration, the tissueto which it is administered, and the physical delivery system in whichthe compound is carried.

The terms “treatment”, “treating”, “treat”, and the like, refer toobtaining a desired pharmacologic and/or physiologic effect. The effectmay be prophylactic in terms of completely or partially preventing adisease or symptom thereof and/or may be therapeutic in terms of apartial or complete cure for a disease and/or adverse effectattributable to the disease. “Treatment” and its grammaticalequivalents, as used herein, covers any treatment of a disease in amammal, particularly in a human, and includes: (a) preventing thedisease from occurring in a subject which may be predisposed to thedisease but has not yet been diagnosed as having it; (b) inhibiting thedisease, i.e., arresting its development or progression; and (c)relieving the disease, i.e., causing regression of the disease and/orrelieving one or more disease symptoms. “Treatment” is also meant toencompass delivery of an agent in order to provide for a pharmacologiceffect, even in the absence of a disease or condition. For example,“treatment” encompasses delivery of a composition that can elicit animmune response or confer immunity in the absence of a diseasecondition, e.g., in the case of a vaccine.

The term “heterologous” when used with reference to portions of anucleic acid or protein indicates that the nucleic acid or proteincomprises two or more subsequences that are not found in the samerelationship to each other in nature. For instance, the nucleic acid istypically recombinantly produced, having two or more sequences fromunrelated genes arranged to make a new functional nucleic acid, e.g., apromoter from one source and a coding region from another source, orcoding regions from different sources. Similarly, a heterologous proteinindicates that the protein comprises two or more subsequences that arenot found in the same relationship to each other in nature (e.g., afusion protein).

The terms “sequence identity,” “percent identity,” and “sequence percentidentity” (or synonyms thereof, e.g., “99% identical”) in the context oftwo or more nucleic acids or polypeptides, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of nucleotides or amino acid residues that are the same, whencompared and aligned (introducing gaps, if necessary) for maximumcorrespondence, not considering any conservative amino acidsubstitutions as part of the sequence identity. The percent identity canbe measured using sequence comparison software or algorithms or byvisual inspection. Various algorithms and software are known in the artthat can be used to obtain alignments of amino acid or nucleotidesequences. Suitable programs to determine percent sequence identityinclude for example the BLAST suite of programs available from the U.S.Government's National Center for Biotechnology Information BLAST website. Comparisons between two sequences can be carried using either theBLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acidsequences, while BLASTP is used to compare amino acid sequences. ALIGN,ALIGN-2 (Genentech, South San Francisco, Calif.) or MegAlign, availablefrom DNASTAR, are additional publicly available software programs thatcan be used to align sequences. One skilled in the art can determineappropriate parameters for maximal alignment by particular alignmentsoftware. In certain embodiments, the default parameters of thealignment software are used.

As used herein, the term “variant” encompasses but is not limited toantibodies or fusion proteins which comprise an amino acid sequencewhich differs from the amino acid sequence of a reference antibody byway of one or more substitutions, deletions and/or additions at certainpositions within or adjacent to the amino acid sequence of the referenceantibody. The variant may comprise one or more conservativesubstitutions in its amino acid sequence as compared to the amino acidsequence of a reference antibody. Conservative substitutions mayinvolve, e.g., the substitution of similarly charged or uncharged aminoacids. The variant retains the ability to specifically bind to theantigen of the reference antibody. The term variant also includespegylated antibodies or proteins.

By “tumor infiltrating lymphocytes” or “TILs” herein is meant apopulation of cells originally obtained as white blood cells that haveleft the bloodstream of a subject and migrated into a tumor. TILsinclude, but are not limited to, CD8+ cytotoxic T cells (lymphocytes),Th1 and Th17 CD4+ T cells, natural killer cells, dendritic cells and M1macrophages. TILs include both primary and secondary TILs. “PrimaryTILs” are those that are obtained from patient tissue samples asoutlined herein (sometimes referred to as “freshly harvested”), and“secondary TILs” are any TIL cell populations that have been expanded orproliferated as discussed herein, including, but not limited to bulkTILs, expanded TILs (“REP TILs”) as well as “reREP TILs” as discussedherein. reREP TILs can include for example second expansion TILs orsecond additional expansion TILs (such as, for example, those describedin Step D of FIG. 27, including TILs referred to as reREP TILs).

TILs can generally be defined either biochemically, using cell surfacemarkers, or functionally, by their ability to infiltrate tumors andeffect treatment. TILs can be generally categorized by expressing one ormore of the following biomarkers: CD4, CD8, TCR αβ, CD27, CD28, CD56,CCR7, CD45Ra, CD95, PD-1, and CD25. Additionally, and alternatively,TILs can be functionally defined by their ability to infiltrate solidtumors upon reintroduction into a patient. TILS may further becharacterized by potency—for example, TILS may be considered potent if,for example, interferon (IFN) release is greater than about 50 pg/mL,greater than about 100 pg/mL, greater than about 150 pg/mL, or greaterthan about 200 pg/mL.

The terms “pharmaceutically acceptable carrier” or “pharmaceuticallyacceptable excipient” are intended to include any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and inert ingredients. The useof such pharmaceutically acceptable carriers or pharmaceuticallyacceptable excipients for active pharmaceutical ingredients is wellknown in the art. Except insofar as any conventional pharmaceuticallyacceptable carrier or pharmaceutically acceptable excipient isincompatible with the active pharmaceutical ingredient, its use in thetherapeutic compositions of the invention is contemplated. Additionalactive pharmaceutical ingredients, such as other drugs, can also beincorporated into the described compositions and methods.

The terms “about” and “approximately” mean within a statisticallymeaningful range of a value. Such a range can be within an order ofmagnitude, preferably within 50%, more preferably within 20%, morepreferably still within 10%, and even more preferably within 5% of agiven value or range. The allowable variation encompassed by the terms“about” or “approximately” depends on the particular system under study,and can be readily appreciated by one of ordinary skill in the art.Moreover, as used herein, the terms “about” and “approximately” meanthat dimensions, sizes, formulations, parameters, shapes and otherquantities and characteristics are not and need not be exact, but may beapproximate and/or larger or smaller, as desired, reflecting tolerances,conversion factors, rounding off, measurement error and the like, andother factors known to those of skill in the art. In general, adimension, size, formulation, parameter, shape or other quantity orcharacteristic is “about” or “approximate” whether or not expresslystated to be such. It is noted that embodiments of very different sizes,shapes and dimensions may employ the described arrangements.

The transitional terms “comprising,” “consisting essentially of,” and“consisting of,” when used in the appended claims, in original andamended form, define the claim scope with respect to what unrecitedadditional claim elements or steps, if any, are excluded from the scopeof the claim(s). The term “comprising” is intended to be inclusive oropen-ended and does not exclude any additional, unrecited element,method, step or material. The term “consisting of” excludes any element,step or material other than those specified in the claim and, in thelatter instance, impurities ordinary associated with the specifiedmaterial(s). The term “consisting essentially of” limits the scope of aclaim to the specified elements, steps or material(s) and those that donot materially affect the basic and novel characteristic(s) of theclaimed invention. All compositions, methods, and kits described hereinthat embody the present invention can, in alternate embodiments, be morespecifically defined by any of the transitional terms “comprising,”“consisting essentially of,” and “consisting of.”

Methods of Treating Cancer

The compositions and methods involving TILs (and populations thereof)described herein can be used in a method for treating hyperproliferativedisorders. In a preferred embodiment, they are for use in treatingcancers. In a preferred embodiment, the invention provides a method oftreating a cancer, wherein the cancer is metastatic melanoma. In apreferred embodiment, the invention provides a method of treating acancer, wherein the cancer is metastatic double-refractory melanoma.

Methods of treating metastatic double-refractory melanoma in accordancewith the present invention include administering to a patient in needthereof a therapeutic population of TILS derived from that patient's owntumor (autologous cell product). In certain embodiments, the cellproduct is composed of ≥90% CD45⁺CD3⁺ T cells. In some embodiments, thecell product is composed of ≥80%, ≥85%, ≥90%, ≥96%, ≥97%, ≥98%, or ≥99%CD45⁺CD3⁺ T cells. Natural killer (NK) cells and B cells may be presentin the cell product, but generally represent less than 5%, less than 4%,less than 3%, less than 2%, or less than 1% of the total cells in thecell product.

For any of the treatment methods described herein, the route ofadministration of TIL therapy is generally by intravenous infusion. Asdescribed in further detail herein, this administration of TILs followstwo or more prior systemic therapies. This administration of TIL therapymay also follow a nonmyeloablative lymphodeletion therapy such ascyclophosphamide and/or fludarbine. In further embodiments, IL-2 isadministered to the patient following the TIL therapy.

As will be appreciated, the TILs used in the treatment methods describedherein can be obtained and processed using methods known in the art anddescribed herein. In certain exemplary embodiments, the therapeuticpopulation TILs used in treatment methods of the invention are expandedfrom tumors resected from the patient with the metastaticdouble-refractory melanoma. Thus, the therapeutic population of TILs is“derived from” TILs from a tumor from the patient. The methods ofexpansion will in further embodiments include expansions such as thosedescribed herein in the section entitled “Methods of Expanding TumorInfiltrating Lymphocytes.” Briefly, such methods include the steps ofresecting a tumor from a patient, the tumor comprising a firstpopulation of TILs, fragmenting the tumor, contacting the tumorfragments with a first cell culture medium to expand that firstpopulation of TILs into a second population of TILs, contacting thesecond population of TILs with a cell culture medium containing IL-2,OKT-3 (anti-CD3 antibody), and irradiated allogeneic peripheral bloodmononuclear cells (PBMCs) to perform an expansion of that secondpopulation of TILs to obtain a third population of TILs, where atherapeutically effective portion of that third population of TILs canbe administered to the patient. In general, the expansion of the secondpopulation into the third (therapeutic) population of cells is performedover a period of 14 days or less. In additional embodiments, methods ofexpanding TILs include those exemplified in co-pending applicationsWO2018/081473, filed Oct. 26, 2017; PCT/US2018/012605, filed Jan. 5,2018; and PCT/US18/12633, filed Jan. 5, 2018, each of which is hereinincorporated by reference in its entirety for all purposes and inparticular for all teachings related to methods of expanding TILs from atumor sample.

In a preferred embodiment, the invention provides a method of treating acancer, wherein the cancer is metastatic double-refractory cutaneousmelanoma.

In a preferred embodiment, the invention provides a method of treating acancer, wherein the cancer is metastatic double-refractory uveal(ocular) melanoma.

As is discussed in further detail herein, the term “double-refractorymelanoma” encompasses melanoma refractory to two or more prior systemictherapies. To be refractory to a prior systemic therapy is meant thatthe patient either had no response or progressed after receiving theprior systemic therapies.

In a preferred embodiment, the invention provides a method of treating acancer, wherein the cancer is metastatic double-refractory melanomarefractory to at least two prior systemic therapies, where those twoprior system therapies are not including neo-adjuvant or adjuvanttherapies such as interferon-α. As will be appreciated, neo-adjuvanttherapies encompass therapies given as a first step to reduce the sizeof a tumor before the main treatment is given. As will further beappreciated, adjuvant therapies include additional cancer treatmentgiven after the primary treatment to lower the risk that the cancer willcome back. The presently disclosed invention comprises TILs treatmentsthat are third, fourth or fifth-line therapies after the melanoma hasnot responded to or has progressed after at least two prior primarytherapies. In further embodiments, the patient has been previouslytreated with one additional prior line of systemic therapy prior toreceiving TILs treatments in accordance with the methods describedherein.

In a preferred embodiment, the invention provides a method of treating acancer, wherein the cancer is metastatic double-refractory melanomarefractory to at least two prior systemic therapies, not includingneo-adjuvant or adjuvant therapies such as interferon-α, wherein priorsystemic therapies containing multiple agents, such as ipilimumab andnivolumab, or concurrent administration of multiple approved orexperimental therapies, are counted as a single prior systemic therapy.In other words, the two prior systemic therapies may include primarytherapies of combination treatments involving two or more therapies thatare considered to be a single therapy. As will be appreciated, thesecombination therapies may include combinations of the same type oftherapies (such as two checkpoint inhibitors), or they may includedifferent types of therapies that are often provided in conjunction as asingle therapy (such as radiation and chemotherapeutics).

In a preferred embodiment, the invention provides a method of treating acancer, wherein the cancer is metastatic double-refractory melanomarefractory to (1) a checkpoint inhibitor and (2) at least one otherprior systemic therapy.

In a preferred embodiment, the invention provides a method of treating acancer, wherein the cancer is metastatic double-refractory melanomarefractory to (1) a BRAF inhibitor and (2) at least one other priorsystemic therapy. In a further embodiment, the at least one other priorsystemic therapy is a checkpoint inhibitor, and in a still furtherembodiment, the checkpoint inhibitor is a PD-1 inhibitor.

In a preferred embodiment, the invention provides a method of treating acancer, wherein the cancer is metastatic double-refractory melanomarefractory to (1) a checkpoint inhibitor and (2) at least one otherprior systemic therapy, wherein the one other prior systemic therapy isa combination of therapies.

In a preferred embodiment, the invention provides a method of treating acancer, wherein the cancer is metastatic double-refractory melanomarefractory to (1) a PD-1 inhibitor and (2) at least one other priorsystemic therapy. In a further embodiment, the patient received no morethan 4 doses of PD-1 inhibitor prior to receiving treatment by TILs inaccordance with the present invention. In a still further embodiment,the patient received no more than 10, 9, 8, 7, 6, 5, 4, 3, or 2 doses ofPD-1 inhibitor receiving treatment by TILs in accordance with thepresent invention.

In a preferred embodiment, the invention provides a method of treating acancer, wherein the cancer is metastatic double-refractory melanomarefractory to (1) a PD-L1 inhibitor and (2) at least one other priorsystemic therapy.

In a preferred embodiment, the invention provides a method of treating acancer, wherein the cancer is metastatic double-refractory melanomarefractory to (1) a CTLA-4 inhibitor and (2) at least one other priorsystemic therapy.

In a preferred embodiment, the invention provides a method of treating acancer, wherein the cancer is metastatic double-refractory melanomarefractory to (1) a PD-1 inhibitor and (2) at least one other priorsystemic therapy, wherein the one other prior systemic therapy is acombination of therapies.

In a preferred embodiment, the invention provides a method of treating acancer, wherein the cancer is metastatic double-refractory melanomarefractory to (1) a PD-L1 inhibitor and (2) at least one other priorsystemic therapy, wherein the one other prior systemic therapy is acombination of therapies.

In a preferred embodiment, the invention provides a method of treating acancer, wherein the cancer is metastatic double-refractory melanomarefractory to (1) a CTLA-4 inhibitor and (2) at least one other priorsystemic therapy, wherein the one other prior systemic therapy is acombination of therapies.

In a preferred embodiment, the invention provides a method of treating acancer, wherein the cancer is metastatic double-refractory melanomarefractory to (1) a checkpoint inhibitor and (2) aldesleukin or abiosimilar or variant thereof, including pegylated IL-2.

In a preferred embodiment, the invention provides a method of treating acancer, wherein the cancer is metastatic double-refractory melanomarefractory to at least two checkpoint inhibitors given as separate priortherapies.

In a preferred embodiment, the invention provides a method of treating acancer, wherein the cancer is metastatic double-refractory melanomarefractory to (1) pembrolizumab, or a biosimilar or variant thereof, and(2) at least one other prior systemic therapy.

In a preferred embodiment, the invention provides a method of treating acancer, wherein the cancer is metastatic double-refractory melanomarefractory to (1) nivolumab, or a biosimilar or variant thereof, and (2)at least one other prior systemic therapy.

In a preferred embodiment, the invention provides a method of treating acancer, wherein the cancer is metastatic double-refractory melanomarefractory to (1) ipilimumab, or a biosimilar or variant thereof, and(2) at least one other prior systemic therapy.

In a preferred embodiment, the invention provides a method of treating acancer, wherein the cancer is metastatic double-refractory melanomarefractory to (1) a combination of nivolumab and ipilimumab, orbiosimilars or variants thereof, and (2) at least one other priorsystemic therapy.

In a preferred embodiment, the invention provides a method of treating acancer, wherein the cancer is metastatic double-refractory melanomarefractory to (1) a checkpoint inhibitor and (2) aldesleukin or abiosimilar or variant thereof, including pegylated IL-2.

In a preferred embodiment, the invention provides a method of treating acancer, wherein the cancer is metastatic double-refractory melanomarefractory to (1) pembrolizumab, or a biosimilar or variant thereof, and(2) at least one other prior systemic therapy, wherein the one otherprior systemic therapy is a combination of therapies.

In a preferred embodiment, the invention provides a method of treating acancer, wherein the cancer is metastatic double-refractory melanomarefractory to (1) nivolumab, or a biosimilar or variant thereof, and (2)at least one other prior systemic therapy, wherein the one other priorsystemic therapy is a combination of therapies.

In a preferred embodiment, the invention provides a method of treating acancer, wherein the cancer is metastatic double-refractory melanomarefractory to (1) ipilimumab, or a biosimilar or variant thereof, and(2) at least one other prior systemic therapy, wherein the one otherprior systemic therapy is a combination of therapies.

In a preferred embodiment, the invention provides a method of treating acancer, wherein the cancer is metastatic double-refractory melanomarefractory to (1) a combination of nivolumab and ipilimumab, orbiosimilars or variants thereof, and (2) at least one other priorsystemic therapy, wherein the one other prior systemic therapy is acombination of therapies.

In any of the foregoing embodiments, the metastatic double-refractorymelanoma may be a cutaneous metastatic double-refractory melanoma.

As is described in further detail herein, the therapeutic population ofTILs used in any of the treatment methods of the invention may becryopreserved or non-cryopreserved. Cryopreserved TILs are producedaccording to methods known in the art or as described in further detailherein.

In accordance with any of the above-described embodiments, thedouble-refractory metastatic melanoma may be refractory to PD-1inhibitors that can include for example antibodies that target PD-1,e.g., but are not limited to nivolumab (BMS-936558, Bristol-MyersSquibb; Opdivo®), pembrolizumab (lambrolizumab, MK03475 or MK-3475,Merck; Keytruda®), humanized anti-PD-1 antibody JS001 (ShangHai JunShi),monoclonal anti-PD-1 antibody TSR-042 (Tesaro, Inc.), Pidilizumab(anti-PD-1 mAb CT-011, Medivation), anti-PD-1 monoclonal AntibodyBGB-A317 (BeiGene), and/or anti-PD-1 antibody SHR-1210 (ShangHaiHengRui), human monoclonal antibody REGN2810 (Regeneron), humanmonoclonal antibody MDX-1106 (Bristol-Myers Squibb), and/or humanizedanti-PD-1 IgG4 antibody PDR001 (Novartis). In some embodiments, the PD-1antibody is from clone: RMP1-14 (rat IgG)-BioXcell cat# BP0146. OtherPD-1 antibodies include those disclosed in U.S. Pat. No. 8,008,449,herein incorporated by reference. In some embodiments, the antibody orantigen-binding portion thereof binds specifically to PD-L1 and inhibitsits interaction with PD-1, thereby increasing immune activity. Anyantibodies known in the art which bind to PD-L1 and disrupt theinteraction between the PD-1 and PD-L1, and stimulates an anti-tumorimmune response, may also be among the systemic therapies to which thedouble-refractory melanoma is refractory. For example, antibodies thattarget PD-L1 and are in clinical trials or are approved and arecommercially available include avelumab (EMD Serono, Pfizer, Bavencio®),durvalumab (MEDI4736, AstraZeneca, Imfinzi®), BMS-936559 (Bristol-MyersSquibb) and atezolizumab (MPDL3280A, Genentech, Tecentriq®). Othersuitable antibodies that target PD-L1 are disclosed in U.S. Pat. No.7,943,743, herein incorporated by reference. It will be understood byone of ordinary skill that any antibody which binds to PD-1 or PD-L1,disrupts the PD-1/PD-L1 interaction, and stimulates an anti-tumor immuneresponse may serve as therapies to which the melanoma treated inaccordance with methods of the present invention are refractory.

Similarly, the melanoma treated in accordance with the methods describedherein may be refractory to BRAF inhibitors, including withoutlimitation inhibitors that affect the BRAF protein directly orinhibitors that affect MEK. BRAF inhibitors include without limitationVemurafenib (Zelboraf®) and dabrafenib (Tafinlar®), as well as GDC-0879,PLX-4720, or sorafenib (Nexavar®). MEK inhibitors include withoutlimitation trametinib (Mekinist®) and cobimetinib (Cotellic®).

In certain embodiments and in accordance with any of the embodimentsdescribed herein, patients treated in accordance with the describedinvention may possess genetic makeups (or have tumors that possessgenetic makeups) that indicate susceptibility or resistance to certaintypes of treatments. For example, patients may show low PDL1 expression,or they may (or may not) possess mutations in the BRAF gene. In specificembodiments, the treatments for double-refractory melanoma describedherein are insensitive/agnostic to the BRAF status (e.g., the presenceor absence of mutations in the BRAF gene). In some embodiments, patientsmay exhibit melanoma resistant to PD-1 or PD-L1 inhibitors. Mechanismsof resistance to PD-1 and PD-L1 inhibitors are known in the art,including resistance based on mutations within genes encoding Januskinase 1 and Janus kinase 2 proteins mutations and resistance based onmutations within genes encoding beta-2-microglobulin, as well as othermutations, which are described, e.g., in Zaretsky, et al., Mutationsassociated with acquired resistance to PD-1 blockade in melanoma, N.Engl. J. Med. 2016, 375, 819-29, the disclosure of which is incorporatedby reference herein.

In accordance with any of the embodiments discussed above, the TILstherapy provided to patients with double-refractory melanoma may includetreatment with therapeutic populations of TILs alone or may include acombination treatment including TILs and one or more other therapies.For example, in some embodiments, the TILs produced as described hereincan be administered in combination with one or more immune checkpointregulators, such as the antibodies described below. For example,antibodies that target PD-1 and which can be co-administered with theTILs of the present invention include, e.g., but are not limited tonivolumab (BMS-936558, Bristol-Myers Squibb; Opdivo®), pembrolizumab(lambrolizumab, MK03475 or MK-3475, Merck; Keytruda®), humanizedanti-PD-1 antibody JS001 (ShangHai JunShi), monoclonal anti-PD-1antibody TSR-042 (Tesaro, Inc.), Pidilizumab (anti-PD-1 mAb CT-011,Medivation), anti-PD-1 monoclonal Antibody BGB-A317 (BeiGene), and/oranti-PD-1 antibody SHR-1210 (ShangHai HengRui), human monoclonalantibody REGN2810 (Regeneron), human monoclonal antibody MDX-1106(Bristol-Myers Squibb), and/or humanized anti-PD-1 IgG4 antibody PDR001(Novartis). In some embodiments, the PD-1 antibody is from clone:RMP1-14 (rat IgG)-BioXcell cat# BP0146. Other suitable antibodiessuitable for use in co-administration methods with TILs producedaccording to Steps A through F as described herein are anti-PD-1antibodies disclosed in U.S. Pat. No. 8,008,449, herein incorporated byreference. In some embodiments, the antibody or antigen-binding portionthereof binds specifically to PD-L1 and inhibits its interaction withPD-1, thereby increasing immune activity. Any antibodies known in theart which bind to PD-L1 and disrupt the interaction between the PD-1 andPD-L1, and stimulates an anti-tumor immune response, are suitable foruse in co-administration methods with TILs produced according to Steps Athrough F as described herein. For example, antibodies that target PD-L1and are in clinical trials or are approved and are commerciallyavailable include avelumab (EMD Serono, Pfizer, Bavencio®), durvalumab(MEDI4736, AstraZeneca, Imfinzi®), BMS-936559 (Bristol-Myers Squibb) andatezolizumab (MPDL3280A, Genentech, Tecentriq®). Other suitableantibodies that target PD-L1 are disclosed in U.S. Pat. No. 7,943,743,herein incorporated by reference. It will be understood by one ofordinary skill that any antibody which binds to PD-1 or PD-L1, disruptsthe PD-1/PD-L1 interaction, and stimulates an anti-tumor immuneresponse, are suitable for use in co-administration methods with TILs.In some embodiments, the patient administered the combination of TILs isco administered with an anti-PD-1 antibody when the patient hasprogressed or had no response to treatment by anti-PD-1 antibody alone.Similarly, TILs therapy may be co-administered with other therapies,such as CTLA-4 inhibitors, BRAF inhibitors, and any other therapiesknown in the art to be useful for treatment of melanoma.

As will be appreciated and in accordance with any of the treatmentmethods described above, any of the additional treatment modalitiesdescribed herein, including BRAF inhibitors, MEK inhibitors, PD-1inhibitors, PD-L1 inhibitors, and CTLA-4 inhibitors, include anyembodiments of such inhibitors as well as any pharmaceuticallyacceptable salt thereof.

In an embodiment, a patient treated with TIL therapies disclosed hereinexhibits an improved response to the response expected from a historicalcontrol, wherein the improved response is determined as overall responserate. In an embodiment, a patient treated with TIL therapies disclosedherein exhibits an improved response to the response expected from ahistorical control, wherein the improved response is determined asoverall response rate, wherein the improvement in overall response rateis at least 5%, at least 10%, at least 15%, at least 20%, at least 25%,or at least 50%. In an embodiment, a patient treated with TIL therapiesdisclosed herein exhibits an improved response to the response expectedfrom a historical control, wherein the improved response is determinedas duration of response. In an embodiment, a patient treated with TILtherapies disclosed herein exhibits an improved response to the responseexpected from a historical control, wherein the improved response isdetermined as duration of response, wherein the improvement in durationof response is at least 5%, at least 10%, at least 15%, at least 20%, atleast 25%, or at least 50%.

Non-Myeloablative Lymphodepletion with Chemotherapy

Experimental findings indicate that lymphodepletion prior to adoptivetransfer of tumor-specific T lymphocytes plays a key role in enhancingtreatment efficacy by eliminating regulatory T cells and competingelements of the immune system (“cytokine sinks”). Accordingly, someembodiments of the invention utilize a lymphodepletion step (sometimesalso referred to as “immunosuppressive conditioning”) on the patientprior to the introduction of the TILs of the invention.

In an embodiment, the invention provides a method of treatingdouble-refractory melanoma with a population of TILs, wherein a patientis pre-treated with non-myeloablative chemotherapy prior to an infusionof TILs. In an embodiment, the non-myeloablative chemotherapy includesone or more chemotherapeutic agents. In an embodiment, thenon-myeloablative chemotherapy is cyclophosphamide 60 mg/kg/d for 2 days(days 27 and 26 prior to TIL infusion) and fludarabine 25 mg/m2/d for 5days (days 27 to 23 prior to TIL infusion). In an embodiment, afternon-myeloablative chemotherapy and TIL infusion (at day 0) according tothe present disclosure, the patient receives an intravenous infusion ofIL-2 intravenously at 720,000 IU/kg every 8 hours to physiologictolerance. In further embodiments, the IL-2 is administered between 3and 24 hours following TIL infusion. In yet further embodiments, theIL-2 is administered following 3-30, 5-25, 7-20, 9-15 hours followingTIL infusion. In still further embodiments, the IL-2 is administered at500,000; 550,000; 600,000; 650,000; 700,000; 750,000; 800,000 IU/kgevery 8-12 hours to physiologic tolerance over 5 days following TILinfusion.

In general, lymphodepletion is achieved using administration offludarabine or cyclophosphamide (the active form being referred to asmafosfamide) and combinations thereof. Such methods are described inGassner, et al., Cancer Immunol. Immunother. 2011, 60, 75-85, Muranski,et al., Nat. Clin. Pract. Oncol., 2006, 3, 668-681, Dudley, et al., J.Clin. Oncol. 2008, 26, 5233-5239, and Dudley, et al., J. Clin. Oncol.2005, 23, 2346-2357, all of which are incorporated by reference hereinin their entireties.

In some embodiments, the fludarabine is administered at a concentrationof 0.5 μg/mL-10 μg/mL fludarabine. In some embodiments, the fludarabineis administered at a concentration of 1 μg/mL fludarabine. In someembodiments, the fludarabine treatment is administered for 1 day, 2days, 3 days, 4 days, 5 days, 6 days, or 7 days or more. In someembodiments, the fludarabine is administered at a dosage of 10mg/kg/day, 15 mg/kg/day, 20 mg/kg/day, 25 mg/kg/day, 30 mg/kg/day, 35mg/kg/day, 40 mg/kg/day, or 45 mg/kg/day. In some embodiments, thefludarabine treatment is administered for 2-7 days at 35 mg/kg/day. Insome embodiments, the fludarabine treatment is administered for 4-5 daysat 35 mg/kg/day. In some embodiments, the fludarabine treatment isadministered for 4-5 days at 25 mg/kg/day.

In some embodiments, the mafosfamide, the active form ofcyclophosphamide, is obtained at a concentration of 0.5 μg/mL-10 μg/mLby administration of cyclophosphamide. In some embodiments, mafosfamide,the active form of cyclophosphamide, is obtained at a concentration of 1μg/mL by administration of cyclophosphamide. In some embodiments, thecyclophosphamide treatment is administered for 1 day, 2 days, 3 days, 4days, 5 days, 6 days, or 7 days or more. In some embodiments, thecyclophosphamide is administered at a dosage of 100 mg/m²/day, 150mg/m²/day, 175 mg/m²/day 200 mg/m²/day, 225 mg/m²/day, 250 mg/m²/day,275 mg/m²/day, or 300 mg/m²/day. In some embodiments, thecyclophosphamide is administered intravenously (i.e., i.v.) In someembodiments, the cyclophosphamide treatment is administered for 2-7 daysat 35 mg/kg/day. In some embodiments, the cyclophosphamide treatment isadministered for 4-5 days at 250 mg/m²/day i.v. In some embodiments, thecyclophosphamide treatment is administered for 4 days at 250 mg/m²/dayi.v.

In some embodiments, lymphodepletion is performed by administering thefludarabine and the cyclophosphamide are together to a patient. In someembodiments, fludarabine is administered at 25 mg/m²/day i.v. andcyclophosphamide is administered at 250 mg/m²/day i.v. over 4 days.

In an embodiment, the lymphodepletion is performed by administration ofcyclophosphamide at a dose of 60 mg/m²/day for two days followed byadministration of fludarabine at a dose of 25 mg/m²/day for five days.

Methods of Expanding Tumor Infiltrating Lymphocytes

An exemplary TIL process known as process 2A containing some of thesefeatures is depicted in FIG. 19, and some of the advantages of thisembodiment of the present invention over process 1C are described inFigures F and G. An embodiment of process 2A is shown FIG. 18.

As discussed herein, the present invention can include a step relatingto the restimulation of cryopreserved TILs to increase their metabolicactivity and thus relative health prior to transplant into a patient,and methods of testing said metabolic health. As generally outlinedherein, TILs are generally taken from a patient sample and manipulatedto expand their number prior to transplant into a patient. In someembodiments, the TILs may be optionally genetically manipulated asdiscussed below.

In some embodiments, the TILs may be cryopreserved. Once thawed, theymay also be restimulated to increase their metabolism prior to infusioninto a patient.

In some embodiments, the first expansion (including processes referredto as the preREP as well as processes shown in FIG. 18 as Step A) isshortened to 3 to 14 days and the second expansion (including processesreferred to as the REP as well as processes shown in FIG. 18 as Step B)is shorted to 7 to 14 days, as discussed in detail below as well as inthe examples and figures. In some embodiments, the first expansion (forexample, an expansion described as Step B in FIG. 18) is shortened to 11days and the second expansion (for example, an expansion as described inStep D in FIG. 18) is shortened to 11 days. In some embodiments, thecombination of the first expansion and second expansion (for example,expansions described as Step B and Step D in FIG. 18) is shortened to 22days, as discussed in detail below and in the examples and figures.

The “Step” Designations A, B, C, etc., below are in reference to FIG. 18and in reference to certain embodiments described herein. The orderingof the Steps below and in FIG. 18 is exemplary and any combination ororder of steps, as well as additional steps, repetition of steps, and/oromission of steps is contemplated by the present application and themethods disclosed herein.

Step A: Obtain Patient Tumor Sample

In general, TILs are initially obtained from a patient tumor sample(“primary TILs”) and then expanded into a larger population for furthermanipulation as described herein, optionally cryopreserved, restimulatedas outlined herein and optionally evaluated for phenotype and metabolicparameters as an indication of TIL health.

A patient tumor sample may be obtained using methods known in the art,generally via surgical resection, needle biopsy or other means forobtaining a sample that contains a mixture of tumor and TIL cells. Ingeneral, the tumor sample may be from any solid tumor, including primarytumors, invasive tumors or metastatic tumors. The tumor sample may alsobe a liquid tumor, such as a tumor obtained from a hematologicalmalignancy. The solid tumor may be of any cancer type, including, butnot limited to, breast, pancreatic, prostate, colorectal, lung, brain,renal, stomach, and skin (including but not limited to squamous cellcarcinoma, basal cell carcinoma, and melanoma). In some embodiments,useful TILs are obtained from malignant melanoma tumors, as these havebeen reported to have particularly high levels of TILs.

The term “solid tumor” refers to an abnormal mass of tissue that usuallydoes not contain cysts or liquid areas. Solid tumors may be benign ormalignant. The term “solid tumor cancer” refers to malignant,neoplastic, or cancerous solid tumors. Solid tumor cancers include, butare not limited to, sarcomas, carcinomas, and lymphomas, such as cancersof the lung, breast, triple negative breast cancer, prostate, colon,rectum, and bladder. In some embodiments, the cancer is selected fromcervical cancer, head and neck cancer (including, for example, head andneck squamous cell carcinoma (HNSCC)) glioblastoma, ovarian cancer,sarcoma, pancreatic cancer, bladder cancer, breast cancer, triplenegative breast cancer, and non-small cell lung carcinoma. The tissuestructure of solid tumors includes interdependent tissue compartmentsincluding the parenchyma (cancer cells) and the supporting stromal cellsin which the cancer cells are dispersed and which may provide asupporting microenvironment.

The term “hematological malignancy” refers to mammalian cancers andtumors of the hematopoietic and lymphoid tissues, including but notlimited to tissues of the blood, bone marrow, lymph nodes, and lymphaticsystem. Hematological malignancies are also referred to as “liquidtumors.” Hematological malignancies include, but are not limited to,acute lymphoblastic leukemia (ALL), chronic lymphocytic lymphoma (CLL),small lymphocytic lymphoma (SLL), acute myelogenous leukemia (AML),chronic myelogenous leukemia (CML), acute monocytic leukemia (AMoL),Hodgkin's lymphoma, and non-Hodgkin's lymphomas. The term “B cellhematological malignancy” refers to hematological malignancies thataffect B cells.

Once obtained, the tumor sample is generally fragmented using sharpdissection into small pieces of between 1 to about 8 mm³, with fromabout 2-3 mm³ being particularly useful. The TILs are cultured fromthese fragments using enzymatic tumor digests. Such tumor digests may beproduced by incubation in enzymatic media (e.g., Roswell Park MemorialInstitute (RPMI) 1640 buffer, 2 mM glutamate, 10 mcg/mL gentamicine, 30units/mL of DNase and 1.0 mg/mL of collagenase) followed by mechanicaldissociation (e.g., using a tissue dissociator). Tumor digests may beproduced by placing the tumor in enzymatic media and mechanicallydissociating the tumor for approximately 1 minute, followed byincubation for 30 minutes at 37° C. in 5% CO₂, followed by repeatedcycles of mechanical dissociation and incubation under the foregoingconditions until only small tissue pieces are present. At the end ofthis process, if the cell suspension contains a large number of redblood cells or dead cells, a density gradient separation using FICOLLbranched hydrophilic polysaccharide may be performed to remove thesecells. Alternative methods known in the art may be used, such as thosedescribed in U.S. Patent Application Publication No. 2012/0244133 A1,the disclosure of which is incorporated by reference herein. Any of theforegoing methods may be used in any of the embodiments described hereinfor methods of expanding TILs or methods treating a cancer.

In general, the harvested cell suspension is called a “primary cellpopulation” or a “freshly harvested” cell population.

In some embodiments, fragmentation includes physical fragmentation,including for example, dissection as well as digestion. In someembodiments, the fragmentation is physical fragmentation. In someembodiments, the fragmentation is dissection. In some embodiments, thefragmentation is by digestion. In some embodiments, TILs can beinitially cultured from enzymatic tumor digests and tumor fragmentsobtained from patients. In an embodiment, TILs can be initially culturedfrom enzymatic tumor digests and tumor fragments obtained from patients.

In some embodiments, where the tumor is a solid tumor, the tumorundergoes physical fragmentation after the tumor sample is obtained in,for example, Step A (as provided in FIG. 18). In some embodiments, thefragmentation occurs before cryopreservation. In some embodiments, thefragmentation occurs after cryopreservation. In some embodiments, thefragmentation occurs after obtaining the tumor and in the absence of anycryopreservation. In some embodiments, the tumor is fragmented and 10,20, 30, 40 or more fragments or pieces are placed in each container forthe first expansion. In some embodiments, the tumor is fragmented and 30or 40 fragments or pieces are placed in each container for the firstexpansion. In some embodiments, the tumor is fragmented and 40 fragmentsor pieces are placed in each container for the first expansion. In someembodiments, the multiple fragments comprise about 4 to about 50fragments, wherein each fragment has a volume of about 27 mm³. In someembodiments, the multiple fragments comprise about 30 to about 60fragments with a total volume of about 1300 mm³ to about 1500 mm³. Insome embodiments, the multiple fragments comprise about 50 fragmentswith a total volume of about 1350 mm³. In some embodiments, the multiplefragments comprise about 50 fragments with a total mass of about 1 gramto about 1.5 grams. In some embodiments, the multiple fragments compriseabout 4 fragments.

In some embodiments, the TILs are obtained from tumor fragments. In someembodiments, the tumor fragment is obtained by sharp dissection. In someembodiments, the tumor fragment is between about 1 mm³ and 10 mm³. Insome embodiments, the tumor fragment is between about 1 mm³ and 8 mm³.In some embodiments, the tumor fragment is about 1 mm³. In someembodiments, the tumor fragment is about 2 mm³. In some embodiments, thetumor fragment is about 3 mm³. In some embodiments, the tumor fragmentis about 4 mm³. In some embodiments, the tumor fragment is about 5 mm³.In some embodiments, the tumor fragment is about 6 mm³. In someembodiments, the tumor fragment is about 7 mm³. In some embodiments, thetumor fragment is about 8 mm³. In some embodiments, the tumor fragmentis about 9 mm³. In some embodiments, the tumor fragment is about 10 mm³.In some embodiments, the tumors are 1-4 mm×1-4 mm×1-4 mm. In someembodiments, the tumors are 1 mm×1 mm×1 mm. In some embodiments, thetumors are 2 mm×2 mm×2 mm. In some embodiments, the tumors are 3 mm×3mm×3 mm. In some embodiments, the tumors are 4 mm×4 mm×4 mm.

In some embodiments, the tumors are resected in order to minimize theamount of hemorrhagic, necrotic, and/or fatty tissues on each piece. Insome embodiments, the tumors are resected in order to minimize theamount of hemorrhagic tissue on each piece. In some embodiments, thetumors are resected in order to minimize the amount of necrotic tissueon each piece. In some embodiments, the tumors are resected in order tominimize the amount of fatty tissue on each piece.

In some embodiments, the tumor fragmentation is performed in order tomaintain the tumor internal structure. In some embodiments, the tumorfragmentation is performed without preforming a sawing motion with ascalpel. In some embodiments, the TILs are obtained from tumor digests.In some embodiments, tumor digests were generated by incubation inenzyme media, for example but not limited to RPMI 1640, 2 mM GlutaMAX,10 mg/mL gentamicin, 30 U/mL DNase, and 1.0 mg/mL collagenase, followedby mechanical dissociation (GentleMACS, Miltenyi Biotec, Auburn,Calif.). After placing the tumor in enzyme media, the tumor can bemechanically dissociated for approximately 1 minute. The solution canthen be incubated for 30 minutes at 37° C. in 5% CO₂ and it thenmechanically disrupted again for approximately 1 minute. After beingincubated again for 30 minutes at 37° C. in 5% CO₂, the tumor can bemechanically disrupted a third time for approximately 1 minute. In someembodiments, after the third mechanical disruption if large pieces oftissue were present, 1 or 2 additional mechanical dissociations wereapplied to the sample, with or without 30 additional minutes ofincubation at 37° C. in 5% CO₂. In some embodiments, at the end of thefinal incubation if the cell suspension contained a large number of redblood cells or dead cells, a density gradient separation using Ficollcan be performed to remove these cells.

In some embodiments, the harvested cell suspension prior to the firstexpansion step is called a “primary cell population” or a “freshlyharvested” cell population.

In some embodiments, cells can be optionally frozen after sample harvestand stored frozen prior to entry into the expansion described in Step B,which is described in further detail below, as well as exemplified inFIG. 18.

Step B: First Expansion

In some embodiments, the present methods provide for obtaining youngTILs, which are capable of increased replication cycles uponadministration to a subject/patient and as such may provide additionaltherapeutic benefits over older TILs (i.e., TILs which have furtherundergone more rounds of replication prior to administration to asubject/patient). Features of young TILs have been described in theliterature, for example Donia, at al., Scandinavian Journal ofImmunology, 75:157-167 (2012); Dudley et al., Clin Cancer Res,16:6122-6131 (2010); Huang et al., J Immunother, 28(3):258-267 (2005);Besser et al., Clin Cancer Res, 19(17):OF1-OF9 (2013); Besser et al., JImmunother 32:415-423 (2009); Robbins, et al., J Immunol 2004;173:7125-7130; Shen et al., J Immunother, 30:123-129 (2007); Zhou, etal., J Immunother, 28:53-62 (2005); and Tran, et al., J Immunother,31:742-751 (2008), all of which are incorporated herein by reference intheir entireties.

The diverse antigen receptors of T and B lymphocytes are produced bysomatic recombination of a limited, but large number of gene segments.These gene segments: V (variable), D (diversity), J (joining), and C(constant), determine the binding specificity and downstreamapplications of immunoglobulins and T-cell receptors (TCRs). The presentinvention provides a method for generating TILs which exhibit andincrease the T-cell repertoire diversity. In some embodiments, the TILsobtained by the present method exhibit an increase in the T-cellrepertoire diversity. In some embodiments, the TILs obtained by thepresent method exhibit an increase in the T-cell repertoire diversity ascompared to freshly harvested TILs and/or TILs prepared using othermethods than those provide herein including for example, methods otherthan those embodied in FIG. 18. In some embodiments, the TILs obtainedby the present method exhibit an increase in the T-cell repertoirediversity as compared to freshly harvested TILs and/or TILs preparedusing methods referred to as process 1C, as exemplified in FIG. 22and/or FIG. 23. In some embodiments, the TILs obtained in the firstexpansion exhibit an increase in the T-cell repertoire diversity. Insome embodiments, the increase in diversity is an increase in theimmunoglobulin diversity and/or the T-cell receptor diversity. In someembodiments, the diversity is in the immunoglobulin is in theimmunoglobulin heavy chain. In some embodiments, the diversity is in theimmunoglobulin is in the immunoglobulin light chain. In someembodiments, the diversity is in the T-cell receptor. In someembodiments, the diversity is in one of the T-cell receptors selectedfrom the group consisting of alpha, beta, gamma, and delta receptors. Insome embodiments, there is an increase in the expression of T-cellreceptor (TCR) alpha and/or beta. In some embodiments, there is anincrease in the expression of T-cell receptor (TCR) alpha. In someembodiments, there is an increase in the expression of T-cell receptor(TCR) beta. In some embodiments, there is an increase in the expressionof TCRab (i.e., TCRα/β).

After dissection or digestion of tumor fragments, for example such asdescribed in Step A of FIG. 18, the resulting cells are cultured inserum containing IL-2 under conditions that favor the growth of TILsover tumor and other cells. In some embodiments, the tumor digests areincubated in 2 mL wells in media comprising inactivated human AB serumwith 6000 IU/mL of IL-2. This primary cell population is cultured for aperiod of days, generally from 3 to 14 days, resulting in a bulk TILpopulation, generally about 1×10⁸ bulk TIL cells. In some embodiments,this primary cell population is cultured for a period of 7 to 14 days,resulting in a bulk TIL population, generally about 1×10⁸ bulk TILcells. In some embodiments, this primary cell population is cultured fora period of 10 to 14 days, resulting in a bulk TIL population, generallyabout 1×10⁸ bulk TIL cells. In some embodiments, this primary cellpopulation is cultured for a period of about 11 days, resulting in abulk TIL population, generally about 1×10⁸ bulk TIL cells.

In a preferred embodiment, expansion of TILs may be performed using aninitial bulk TIL expansion step (for example such as those described inStep B of FIG. 18, which can include processes referred to as pre-REP)as described below and herein, followed by a second expansion (Step D,including processes referred to as rapid expansion protocol (REP) steps)as described below under Step D and herein, followed by optionalcryopreservation, and followed by a second Step D (including processesreferred to as restimulation REP steps) as described below and herein.The TILs obtained from this process may be optionally characterized forphenotypic characteristics and metabolic parameters as described herein.

In embodiments where TIL cultures are initiated in 24-well plates, forexample, using Costar 24-well cell culture cluster, flat bottom (CorningIncorporated, Corning, N.Y., each well can be seeded with 1×10⁶ tumordigest cells or one tumor fragment in 2 mL of complete medium (CM) withIL-2 (6000 IU/mL; Chiron Corp., Emeryville, Calif.). In someembodiments, the tumor fragment is between about 1 mm³ and 10 mm³.

In some embodiments, the first expansion culture medium is referred toas “CM”, an abbreviation for culture media. In some embodiments, CM forStep B consists of RPMI 1640 with GlutaMAX, supplemented with 10% humanAB serum, 25 mM Hepes, and 10 mg/mL gentamicin. In embodiments wherecultures are initiated in gas-permeable flasks with a 40 mL capacity anda 10 cm² gas-permeable silicon bottom (for example, G-Rex10; Wilson WolfManufacturing, New Brighton, Minn.) (FIG. 1), each flask was loaded with10-40×10⁶ viable tumor digest cells or 5-30 tumor fragments in 10-40 mLof CM with IL-2. Both the G-Rex10 and 24-well plates were incubated in ahumidified incubator at 37° C. in 5% CO₂ and 5 days after cultureinitiation, half the media was removed and replaced with fresh CM andIL-2 and after day 5, half the media was changed every 2-3 days.

After preparation of the tumor fragments, the resulting cells (i.e.,fragments) are cultured in serum containing IL-2 under conditions thatfavor the growth of TILs over tumor and other cells. In someembodiments, the tumor digests are incubated in 2 mL wells in mediacomprising inactivated human AB serum (or, in some cases, as outlinedherein, in the presence of aAPC cell population) with 6000 IU/mL ofIL-2. This primary cell population is cultured for a period of days,generally from 10 to 14 days, resulting in a bulk TIL population,generally about 1×10⁸ bulk TIL cells. In some embodiments, the growthmedia during the first expansion comprises IL-2 or a variant thereof. Insome embodiments, the IL is recombinant human IL-2 (rhIL-2). In someembodiments the IL-2 stock solution has a specific activity of 20-30×10⁶IU/mg for a 1 mg vial. In some embodiments the IL-2 stock solution has aspecific activity of 20×10⁶ IU/mg for a 1 mg vial. In some embodimentsthe IL-2 stock solution has a specific activity of 25×10⁶ IU/mg for a 1mg vial. In some embodiments the IL-2 stock solution has a specificactivity of 30×10⁶ IU/mg for a 1 mg vial. In some embodiments, the IL-2stock solution has a final concentration of 4-8×10⁶ IU/mg of IL-2. Insome embodiments, the IL-2 stock solution has a final concentration of5-7×10⁶ IU/mg of IL-2. In some embodiments, the IL-2 stock solution hasa final concentration of 6×10⁶ IU/mg of IL-2. In some embodiments, theIL-2 stock solution is prepare as described in Example 9. In someembodiments, the first expansion culture media comprises about 10,000IU/mL of IL-2, about 9,000 IU/mL of IL-2, about 8,000 IU/mL of IL-2,about 7,000 IU/mL of IL-2, about 6000 IU/mL of IL-2 or about 5,000 IU/mLof IL-2. In some embodiments, the first expansion culture mediacomprises about 9,000 IU/mL of IL-2 to about 5,000 IU/mL of IL-2. Insome embodiments, the first expansion culture media comprises about8,000 IU/mL of IL-2 to about 6,000 IU/mL of IL-2. In some embodiments,the first expansion culture media comprises about 7,000 IU/mL of IL-2 toabout 6,000 IU/mL of IL-2. In some embodiments, the first expansionculture media comprises about 6,000 IU/mL of IL-2. In an embodiment, thecell culture medium further comprises IL-2. In some embodiments, thecell culture medium comprises about 3000 IU/mL of IL-2. In anembodiment, the cell culture medium further comprises IL-2. In apreferred embodiment, the cell culture medium comprises about 3000 IU/mLof IL-2. In an embodiment, the cell culture medium comprises about 1000IU/mL, about 1500 IU/mL, about 2000 IU/mL, about 2500 IU/mL, about 3000IU/mL, about 3500 IU/mL, about 4000 IU/mL, about 4500 IU/mL, about 5000IU/mL, about 5500 IU/mL, about 6000 IU/mL, about 6500 IU/mL, about 7000IU/mL, about 7500 IU/mL, or about 8000 IU/mL of IL-2. In an embodiment,the cell culture medium comprises between 1000 and 2000 IU/mL, between2000 and 3000 IU/mL, between 3000 and 4000 IU/mL, between 4000 and 5000IU/mL, between 5000 and 6000 IU/mL, between 6000 and 7000 IU/mL, between7000 and 8000 IU/mL, or about 8000 IU/mL of IL-2.

In some embodiments, first expansion culture media comprises about 500IU/mL of IL-15, about 400 IU/mL of IL-15, about 300 IU/mL of IL-15,about 200 IU/mL of IL-15, about 180 IU/mL of IL-15, about 160 IU/mL ofIL-15, about 140 IU/mL of IL-15, about 120 IU/mL of IL-15, or about 100IU/mL of IL-15. In some embodiments, the first expansion culture mediacomprises about 500 IU/mL of IL-15 to about 100 IU/mL of IL-15. In someembodiments, the first expansion culture media comprises about 400 IU/mLof IL-15 to about 100 IU/mL of IL-15. In some embodiments, the firstexpansion culture media comprises about 300 IU/mL of IL-15 to about 100IU/mL of IL-15. In some embodiments, the first expansion culture mediacomprises about 200 IU/mL of IL-15. In some embodiments, the cellculture medium comprises about 180 IU/mL of IL-15. In an embodiment, thecell culture medium further comprises IL-15. In a preferred embodiment,the cell culture medium comprises about 180 IU/mL of IL-15.

In some embodiments, first expansion culture media comprises about 20IU/mL of IL-21, about 15 IU/mL of IL-21, about 12 IU/mL of IL-21, about10 IU/mL of IL-21, about 5 IU/mL of IL-21, about 4 IU/mL of IL-21, about3 IU/mL of IL-21, about 2 IU/mL of IL-21, about 1 IU/mL of IL-21, orabout 0.5 IU/mL of IL-21. In some embodiments, the first expansionculture media comprises about 20 IU/mL of IL-21 to about 0.5 IU/mL ofIL-21. In some embodiments, the first expansion culture media comprisesabout 15 IU/mL of IL-21 to about 0.5 IU/mL of IL-21. In someembodiments, the first expansion culture media comprises about 12 IU/mLof IL-21 to about 0.5 IU/mL of IL-21. In some embodiments, the firstexpansion culture media comprises about 10 IU/mL of IL-21 to about 0.5IU/mL of IL-21. In some embodiments, the first expansion culture mediacomprises about 5 IU/mL of IL-21 to about 1 IU/mL of IL-21. In someembodiments, the first expansion culture media comprises about 2 IU/mLof IL-21. In some embodiments, the cell culture medium comprises about 1IU/mL of IL-21. In some embodiments, the cell culture medium comprisesabout 0.5 IU/mL of IL-21. In an embodiment, the cell culture mediumfurther comprises IL-21. In a preferred embodiment, the cell culturemedium comprises about 1 IU/mL of IL-21.

In an embodiment, the cell culture medium comprises OKT-3 antibody. Insome embodiments, the cell culture medium comprises about 30 ng/mL ofOKT-3 antibody. In some embodiments, the cell culture medium comprisesabout 15 ng/mL of OKT-3 antibody. In an embodiment, the cell culturemedium comprises about 0.1 ng/mL, about 0.5 ng/mL, about 1 ng/mL, about2.5 ng/mL, about 5 ng/mL, about 7.5 ng/mL, about 10 ng/mL, about 15ng/mL, about 20 ng/mL, about 25 ng/mL, about 30 ng/mL, about 35 ng/mL,about 40 ng/mL, about 50 ng/mL, about 60 ng/mL, about 70 ng/mL, about 80ng/mL, about 90 ng/mL, about 100 ng/mL, about 200 ng/mL, about 500ng/mL, and about 1 μg/mL of OKT-3 antibody. In an embodiment, the cellculture medium comprises between 0.1 ng/mL and 1 ng/mL, between 1 ng/mLand 5 ng/mL, between 5 ng/mL and 10 ng/mL, between 10 ng/mL and 20ng/mL, between 20 ng/mL and 30 ng/mL, between 30 ng/mL and 40 ng/mL,between 40 ng/mL and 50 ng/mL, and between 50 ng/mL and 100 ng/mL ofOKT-3 antibody. In some embodiments, the cell culture medium does notcomprise OKT-3 antibody. In some embodiments, the OKT-3 antibody ismuromonab.

TABLE 3 Amino acid sequences of muromonab (exemplary OKT-3 antibody)Identifier Sequence (One-Letter Amino Acid Symbols) SEQ ID NO: 1QVQLQQSGAE LARPGASVKM SCKASGYTFT RYTMHWVKQR PGQGLEWIGY INPSRGYTNY 60Muromonab heavy NQHFKDKATL TTDKSSSTAY MQLSSLTSED SAVYYCARYY DDHYCLDYWGQGTTLTVSSA 120 chain ETTAPSVYPL APVCGGTTGS SVTLGCLVKG YFPEPVTLTWNSGSLSSGVH TFPAVLQSDL 180 YTLSSSVTVT SSTWPSQSIT CNVAHPASST EVDKKIEPRPKSCDKTHTCP PCPAPELLGG 240 PSVFLFPPKP EDTLMISRTP EVTCVVVDVS HEDPEVKFNWYVDGVEVHNA ETKPREEQYN 300 STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTISKARGQPREPQ VYTLPPSRDE 360 LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPVLDSDGSFFLY SKLTVDKSRW 420 QQGNVFSCSV MHEALHNHYT QKSLSLSPGK 450 SEQ IDNO: 2 QIVLTQSPAI MSASPGEKVT MTCSASSSVS YMNWYQQKSG TSPKRWIYDT SKLASGVPAH60 Muromonab light FRGSGSGTSY SLTISGMEAE DAATYYCQQW SSNPFTFGSGTKLEINRADT APTVSIFPPS 120 chain SEQLTSGGAS VVCFLNNFYP KDINVKWKIDGSERQNGVLN SWTDQDSKDS TYSMSSTLTL 180 TKDEYERHNS YTCEATHKTS TSPIVKSFNRNEC 213

In some embodiments, the cell culture medium comprises one or moreTNFRSF agonists in a cell culture medium. In some embodiments, theTNFRSF agonist comprises a 4-1BB agonist. In some embodiments, theTNFRSF agonist is a 4-1BB agonist, and the 4-1BB agonist is selectedfrom the group consisting of urelumab, utomilumab, EU-101, a fusionprotein, and fragments, derivatives, variants, biosimilars, andcombinations thereof. In some embodiments, the TNFRSF agonist is addedat a concentration sufficient to achieve a concentration in the cellculture medium of between 0.1 μg/mL and 100 μg/mL. In some embodiments,the TNFRSF agonist is added at a concentration sufficient to achieve aconcentration in the cell culture medium of between 20 μg/mL and 40μg/mL.

In some embodiments, in addition to one or more TNFRSF agonists, thecell culture medium further comprises IL-2 at an initial concentrationof about 3000 IU/mL and OKT-3 antibody at an initial concentration ofabout 30 ng/mL, and wherein the one or more TNFRSF agonists comprises a4-1BB agonist.

In some embodiments, the first expansion culture medium is referred toas “CM”, an abbreviation for culture media. In some embodiments, it isreferred to as CM1 (culture medium 1). In some embodiments, CM consistsof RPMI 1640 with GlutaMAX, supplemented with 10% human AB serum, 25 mMHepes, and 10 mg/mL gentamicin. In embodiments where cultures areinitiated in gas-permeable flasks with a 40 mL capacity and a 10 cm²gas-permeable silicon bottom (for example, G-Rex10; Wilson WolfManufacturing, New Brighton, Minn.) (FIG. 1), each flask was loaded with10-40×10⁶ viable tumor digest cells or 5-30 tumor fragments in 10-40 mLof CM with IL-2. Both the G-Rex10 and 24-well plates were incubated in ahumidified incubator at 37° C. in 5% CO₂ and 5 days after cultureinitiation, half the media was removed and replaced with fresh CM andIL-2 and after day 5, half the media was changed every 2-3 days. In someembodiments, the CM is the CM1 described in the Examples. In someembodiments, the first expansion occurs in an initial cell culturemedium or a first cell culture medium. In some embodiments, the initialcell culture medium or the first cell culture medium comprises IL-2.

In some embodiments, the first expansion (including processes such asfor example those described in Step B of FIG. 18, which can includethose sometimes referred to as the pre-REP) process is shortened to 3-14days, as discussed in the examples and figures. In some embodiments, thefirst expansion (including processes such as for example those describedin Step B of FIG. 18, which can include those sometimes referred to asthe pre-REP) is shortened to 7 to 14 days, as discussed in the Examplesand shown in FIGS. 4 and 5, as well as including for example, anexpansion as described in Step B of FIG. 18. In some embodiments, thefirst expansion of Step B is shortened to 10-14 days. In someembodiments, the first expansion is shortened to 11 days, as discussedin, for example, an expansion as described in Step B of FIG. 18.

In some embodiments, the first TIL expansion can proceed for 1 day, 2days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days,11 days, 12 days, 13 days, or 14 days. In some embodiments, the firstTIL expansion can proceed for 1 day to 14 days. In some embodiments, thefirst TIL expansion can proceed for 2 days to 14 days. In someembodiments, the first TIL expansion can proceed for 3 days to 14 days.In some embodiments, the first TIL expansion can proceed for 4 days to14 days. In some embodiments, the first TIL expansion can proceed for 5days to 14 days. In some embodiments, the first TIL expansion canproceed for 6 days to 14 days. In some embodiments, the first TILexpansion can proceed for 7 days to 14 days. In some embodiments, thefirst TIL expansion can proceed for 8 days to 14 days. In someembodiments, the first TIL expansion can proceed for 9 days to 14 days.In some embodiments, the first TIL expansion can proceed for 10 days to14 days. In some embodiments, the first TIL expansion can proceed for 11days to 14 days. In some embodiments, the first TIL expansion canproceed for 12 days to 14 days. In some embodiments, the first TILexpansion can proceed for 13 days to 14 days. In some embodiments, thefirst TIL expansion can proceed for 14 days. In some embodiments, thefirst TIL expansion can proceed for 1 day to 11 days. In someembodiments, the first TIL expansion can proceed for 2 days to 11 days.In some embodiments, the first TIL expansion can proceed for 3 days to11 days. In some embodiments, the first TIL expansion can proceed for 4days to 11 days. In some embodiments, the first TIL expansion canproceed for 5 days to 11 days. In some embodiments, the first TILexpansion can proceed for 6 days to 11 days. In some embodiments, thefirst TIL expansion can proceed for 7 days to 11 days. In someembodiments, the first TIL expansion can proceed for 8 days to 11 days.In some embodiments, the first TIL expansion can proceed for 9 days to11 days. In some embodiments, the first TIL expansion can proceed for 10days to 11 days. In some embodiments, the first TIL expansion canproceed for 11 days.

In some embodiments, a combination of IL-2, IL-7, IL-15, and/or IL-21are employed as a combination during the first expansion. In someembodiments, IL-2, IL-7, IL-15, and/or IL-21 as well as any combinationsthereof can be included during the first expansion, including forexample during a Step B processes according to FIG. 18, as well asdescribed herein. In some embodiments, a combination of IL-2, IL-15, andIL-21 are employed as a combination during the first expansion. In someembodiments, IL-2, IL-15, and IL-21 as well as any combinations thereofcan be included during Step B processes according to FIG. 18 and asdescribed herein.

In some embodiments, the first expansion (including processes referredto as the pre-REP; for example, Step B according to FIG. 18) process isshortened to 3 to 14 days, as discussed in the examples and figures. Insome embodiments, the first expansion of Step B is shortened to 7 to 14days. In some embodiments, the first expansion of Step B is shortened to10 to14 days. In some embodiments, the first expansion is shortened to11 days.

In some embodiments, the first expansion, for example, Step B accordingto FIG. 18, is performed in a closed system bioreactor. In someembodiments, a closed system is employed for the TIL expansion, asdescribed herein. In some embodiments, a single bioreactor is employed.In some embodiments, the single bioreactor employed is for example aG-REX-10 or a G-REX-100. In some embodiments, the closed systembioreactor is a single bioreactor.

Step C: First Expansion to Second Expansion Transition

In some cases, the bulk TIL population obtained from the firstexpansion, including for example the TIL population obtained from forexample, Step B as indicated in FIG. 18, can be cryopreservedimmediately, using the protocols discussed herein below. Alternatively,the TIL population obtained from the first expansion, referred to as thesecond TIL population, can be subjected to a second expansion (which caninclude expansions sometimes referred to as REP) and then cryopreservedas discussed below. Similarly, in the case where genetically modifiedTILs will be used in therapy, the first TIL population (sometimesreferred to as the bulk TIL population) or the second TIL population(which can in some embodiments include populations referred to as theREP TIL populations) can be subjected to genetic modifications forsuitable treatments prior to expansion or after the first expansion andprior to the second expansion.

In some embodiments, the TILs obtained from the first expansion (forexample, from Step B as indicated in FIG. 18) are stored untilphenotyped for selection. In some embodiments, the TILs obtained fromthe first expansion (for example, from Step B as indicated in FIG. 18)are not stored and proceed directly to the second expansion. In someembodiments, the TILs obtained from the first expansion are notcryopreserved after the first expansion and prior to the secondexpansion. In some embodiments, the transition from the first expansionto the second expansion occurs at about 3 days, 4, days, 5 days, 6 days,7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 daysfrom when fragmentation occurs. In some embodiments, the transition fromthe first expansion to the second expansion occurs at about 3 days to 14days from when fragmentation occurs. In some embodiments, the transitionfrom the first expansion to the second expansion occurs at about 4 daysto 14 days from when fragmentation occurs. In some embodiments, thetransition from the first expansion to the second expansion occurs atabout 4 days to 10 days from when fragmentation occurs. In someembodiments, the transition from the first expansion to the secondexpansion occurs at about 7 days to 14 days from when fragmentationoccurs. In some embodiments, the transition from the first expansion tothe second expansion occurs at about 14 days from when fragmentationoccurs.

In some embodiments, the transition from the first expansion to thesecond expansion occurs at 1 day, 2 days, 3 days, 4 days, 5 days, 6days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14days from when fragmentation occurs. In some embodiments, the transitionfrom the first expansion to the second expansion occurs 1 day to 14 daysfrom when fragmentation occurs. In some embodiments, the first TILexpansion can proceed for 2 days to 14 days. In some embodiments, thetransition from the first expansion to the second expansion occurs 3days to 14 days from when fragmentation occurs. In some embodiments, thetransition from the first expansion to the second expansion occurs 4days to 14 days from when fragmentation occurs. In some embodiments, thetransition from the first expansion to the second expansion occurs 5days to 14 days from when fragmentation occurs. In some embodiments, thetransition from the first expansion to the second expansion occurs 6days to 14 days from when fragmentation occurs. In some embodiments, thetransition from the first expansion to the second expansion occurs 7days to 14 days from when fragmentation occurs. In some embodiments, thetransition from the first expansion to the second expansion occurs 8days to 14 days from when fragmentation occurs. In some embodiments, thetransition from the first expansion to the second expansion occurs 9days to 14 days from when fragmentation occurs. In some embodiments, thetransition from the first expansion to the second expansion occurs 10days to 14 days from when fragmentation occurs. In some embodiments, thetransition from the first expansion to the second expansion occurs 11days to 14 days from when fragmentation occurs. In some embodiments, thetransition from the first expansion to the second expansion occurs 12days to 14 days from when fragmentation occurs. In some embodiments, thetransition from the first expansion to the second expansion occurs 13days to 14 days from when fragmentation occurs. In some embodiments, thetransition from the first expansion to the second expansion occurs 14days from when fragmentation occurs. In some embodiments, the transitionfrom the first expansion to the second expansion occurs 1 day to 11 daysfrom when fragmentation occurs. In some embodiments, the transition fromthe first expansion to the second expansion occurs 2 days to 11 daysfrom when fragmentation occurs. In some embodiments, the transition fromthe first expansion to the second expansion occurs 3 days to 11 daysfrom when fragmentation occurs. In some embodiments, the transition fromthe first expansion to the second expansion occurs 4 days to 11 daysfrom when fragmentation occurs. In some embodiments, the transition fromthe first expansion to the second expansion occurs 5 days to 11 daysfrom when fragmentation occurs. In some embodiments, the transition fromthe first expansion to the second expansion occurs 6 days to 11 daysfrom when fragmentation occurs. In some embodiments, the transition fromthe first expansion to the second expansion occurs 7 days to 11 daysfrom when fragmentation occurs. In some embodiments, the transition fromthe first expansion to the second expansion occurs 8 days to 11 daysfrom when fragmentation occurs. In some embodiments, the transition fromthe first expansion to the second expansion occurs 9 days to 11 daysfrom when fragmentation occurs. In some embodiments, the transition fromthe first expansion to the second expansion occurs 10 days to 11 daysfrom when fragmentation occurs. In some embodiments, the transition fromthe first expansion to the second expansion occurs 11 days from whenfragmentation occurs.

In some embodiments, the TILs are not stored after the first expansionand prior to the second expansion, and the TILs proceed directly to thesecond expansion (for example, in some embodiments, there is no storageduring the transition from Step B to Step D as shown in FIG. 18). Insome embodiments, the transition occurs in closed system, as describedherein. In some embodiments, the TILs from the first expansion, thesecond population of TILs, proceeds directly into the second expansionwith no transition period.

In some embodiments, the transition from the first expansion to thesecond expansion, for example, Step C according to FIG. 18, is performedin a closed system bioreactor. In some embodiments, a closed system isemployed for the TIL expansion, as described herein. In someembodiments, a single bioreactor is employed. In some embodiments, thesingle bioreactor employed is for example a G-REX-10 or a G-REX-100. Insome embodiments, the closed system bioreactor is a single bioreactor.

Cytokines

The expansion methods described herein generally use culture media withhigh doses of a cytokine, in particular IL-2, as is known in the art.

Alternatively, using combinations of cytokines for the rapid expansionand/or second expansion of TILS is additionally possible, withcombinations of two or more of IL-2, IL-15 and IL-21 as is generallyoutlined in International Publication No. WO 2015/189356 andInternational Publication No. WO 2015/189357, hereby expresslyincorporated by reference in their entirety for all purposes and inparticular for all teachings related to use of cytokines in cellexpansion methods. Thus, possible combinations include IL-2 and IL-15,IL-2 and IL-21, IL-15 and IL-21 and IL-2, IL-15 and IL-21, with thelatter finding particular use in many embodiments. The use ofcombinations of cytokines specifically favors the generation oflymphocytes, and in particular T-cells as described therein.

TABLE 4 Amino acid sequences of interleukins. Identifier Sequence(One-Letter Amino Acid Symbols) SEQ ID NO: 3 MAPTSSSTEK TQLQLEHLLLDLQMILNGIN NYKNPKLTRM LTFKFYMPKK ATELKHLQCL 60 recombinant EEELIKPLEEVLNLAQSENFH LRPRDLISNI NVIVLELKGS ETTFMCEYAD ETATIVEFLN 120 human IL-2RWITFCQSII STLT 134 (rhIL-2) SEQ ID NO: 4 PTSSSTKKTQ LQLEHLLLDLQMILNGINNY KNPKLTRMLT FKFYMPKKAT ELKHLQCLEE 60 Aldesleukin ELKPLEEVLNLAQSKNFHLR PRDLISNINV IVLELKGSET TFMCEYADET ATIVEFLNRW 120 ITFSQSIIST LT132 SEQ ID NO: 5 MHKCDITLQE IIKTLNSLTE QKTLCTELTV TDIFAASENT TEKETFCRAATVLRQFYSHH 60 recombinant EXDTRCLGAT AQQFHRHKQL IRFLKRLDRN LWGLAGLNSCPVKEANQSTL ENFLERLKTI 120 human IL-4 MREKYSKCSS 130 (rhIL-4) SEQ ID NO:6 MDCDIEGEDG EQYESVLMVS IDQLLDSMKE IGSNCLNNEF NFFERHICDA NKEGMFLFRA 60recombinant ARKLRQFLEM NSTGDFDLHL LKVSEGTTIL LNCTGQVKGR KPAALGEAQPTKSLEENKSL 120 human IL-7 KEQKKLNDLC FLKRLLQEIK TCWNKILMGT KEH 153(rhIL-7) SEQ ID NO: 7 MNWVNVISDL KKIEDLIQSM HIDATLYTES DVHPSCKVTAMKCFLLELQV ISLESGDASI 60 recombinant HDTVENLIIL ANNSLSSNGN VTESGCKECEELEEKNIKEF LQSFVHIVQM FINTS 115 human IL-15 (rhIL-15) SEQ ID NO: 8MQDRHMIRMR QLIDIVDQLK NYVNDLVPEF LPAPEDVETN CEWSAFSCFQ KAQLKSANTG 60recombinant NNERIINVSI KKLKRKPPST NAGRRQKHRL TCPSCDSYEK KPPKEFLERFKSLLQKMIHQ 120 human IL-21 HLSSRTHGSE DS 132 (rhIL-21)

Step D: Second Expansion

In some embodiments, the TIL cell population is expanded in number afterharvest and initial bulk processing for example, after Step A and StepB, and the transition referred to as Step C, as indicated in FIG. 18).This further expansion is referred to herein as the second expansion,which can include expansion processes generally referred to in the artas a rapid expansion process (REP; as well as processes as indicated inStep D of FIG. 18). The second expansion is generally accomplished usinga culture media comprising a number of components, including feedercells, a cytokine source, and an anti-CD3 antibody, in a gas-permeablecontainer.

In some embodiments, the second expansion or second TIL expansion (whichcan include expansions sometimes referred to as REP; as well asprocesses as indicated in Step D of FIG. 18) of TIL can be performedusing any TIL flasks or containers known by those of skill in the art.In some embodiments, the second TIL expansion can proceed for 7 days, 8days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days. In someembodiments, the second TIL expansion can proceed for about 7 days toabout 14 days. In some embodiments, the second TIL expansion can proceedfor about 8 days to about 14 days. In some embodiments, the second TILexpansion can proceed for about 9 days to about 14 days. In someembodiments, the second TIL expansion can proceed for about 10 days toabout 14 days. In some embodiments, the second TIL expansion can proceedfor about 11 days to about 14 days. In some embodiments, the second TILexpansion can proceed for about 12 days to about 14 days. In someembodiments, the second TIL expansion can proceed for about 13 days toabout 14 days. In some embodiments, the second TIL expansion can proceedfor about 14 days.

In an embodiment, the second expansion can be performed in a gaspermeable container using the methods of the present disclosure(including for example, expansions referred to as REP; as well asprocesses as indicated in Step D of FIG. 18). For example, TILs can berapidly expanded using non-specific T-cell receptor stimulation in thepresence of interleukin-2 (IL-2) or interleukin-15 (IL-15). Thenon-specific T-cell receptor stimulus can include, for example, ananti-CD3 antibody, such as about 30 ng/ml of OKT3, a mouse monoclonalanti-CD3 antibody (commercially available from Ortho-McNeil, Raritan,N.J. or Miltenyi Biotech, Auburn, Calif.) or UHCT-1 (commerciallyavailable from BioLegend, San Diego, Calif., USA). TILs can be expandedto induce further stimulation of the TILs in vitro by including one ormore antigens during the second expansion, including antigenic portionsthereof, such as epitope(s), of the cancer, which can be optionallyexpressed from a vector, such as a human leukocyte antigen A2 (HLA-A2)binding peptide, e.g., 0.3 μM MART-1:26-35 (27 L) or gpl 00:209-217(210M), optionally in the presence of a T-cell growth factor, such as300 IU/mL IL-2 or IL-15. Other suitable antigens may include, e.g.,NY-ESO-1, TRP-1, TRP-2, tyrosinase cancer antigen, MAGE-A3, SSX-2, andVEGFR2, or antigenic portions thereof. TIL may also be rapidly expandedby re-stimulation with the same antigen(s) of the cancer pulsed ontoHLA-A2-expressing antigen-presenting cells. Alternatively, the TILs canbe further re-stimulated with, e.g., example, irradiated, autologouslymphocytes or with irradiated HLA-A2+ allogeneic lymphocytes and IL-2.In some embodiments, the re-stimulation occurs as part of the secondexpansion. In some embodiments, the second expansion occurs in thepresence of irradiated, autologous lymphocytes or with irradiatedHLA-A2+ allogeneic lymphocytes and IL-2.

In an embodiment, the cell culture medium for the second expansion stepfurther comprises IL-2. In some embodiments, the cell culture mediumcomprises about 3000 IU/mL of IL-2. In an embodiment, the cell culturemedium comprises about 1000 IU/mL, about 1500 IU/mL, about 2000 IU/mL,about 2500 IU/mL, about 3000 IU/mL, about 3500 IU/mL, about 4000 IU/mL,about 4500 IU/mL, about 5000 IU/mL, about 5500 IU/mL, about 6000 IU/mL,about 6500 IU/mL, about 7000 IU/mL, about 7500 IU/mL, or about 8000IU/mL of IL-2. In an embodiment, the cell culture medium comprisesbetween 1000 and 2000 IU/mL, between 2000 and 3000 IU/mL, between 3000and 4000 IU/mL, between 4000 and 5000 IU/mL, between 5000 and 6000IU/mL, between 6000 and 7000 IU/mL, between 7000 and 8000 IU/mL, orbetween 8000 IU/mL of IL-2.

In an embodiment, the cell culture medium comprises OKT-3 antibody. Insome embodiments, the cell culture medium comprises about 30 ng/mL ofOKT-3 antibody. In an embodiment, the cell culture medium comprisesabout 0.1 ng/mL, about 0.5 ng/mL, about 1 ng/mL, about 2.5 ng/mL, about5 ng/mL, about 7.5 ng/mL, about 10 ng/mL, about 15 ng/mL, about 20ng/mL, about 25 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL,about 50 ng/mL, about 60 ng/mL, about 70 ng/mL, about 80 ng/mL, about 90ng/mL, about 100 ng/mL, about 200 ng/mL, about 500 ng/mL, and about 1pg/mL of OKT-3 antibody. In an embodiment, the cell culture mediumcomprises between 0.1 ng/mL and 1 ng/mL, between 1 ng/mL and 5 ng/mL,between 5 ng/mL and 10 ng/mL, between 10 ng/mL and 20 ng/mL, between 20ng/mL and 30 ng/mL, between 30 ng/mL and 40 ng/mL, between 40 ng/mL and50 ng/mL, and between 50 ng/mL and 100 ng/mL of OKT-3 antibody. In someembodiments, the cell culture medium does not comprise OKT-3 antibody.In some embodiments, the OKT-3 antibody is muromonab.

In some embodiments, the cell culture medium comprises one or moreTNFRSF agonists in a cell culture medium. In some embodiments, theTNFRSF agonist comprises a 4-1BB agonist. In some embodiments, theTNFRSF agonist is a 4-1BB agonist, and the 4-1BB agonist is selectedfrom the group consisting of urelumab, utomilumab, EU-101, a fusionprotein, and fragments, derivatives, variants, biosimilars, andcombinations thereof. In some embodiments, the TNFRSF agonist is addedat a concentration sufficient to achieve a concentration in the cellculture medium of between 0.1 pg/mL and 100 pg/mL. In some embodiments,the TNFRSF agonist is added at a concentration sufficient to achieve aconcentration in the cell culture medium of between 20 pg/mL and 40pg/mL.

In some embodiments, in addition to one or more TNFRSF agonists, thecell culture medium further comprises IL-2 at an initial concentrationof about 3000 IU/mL and OKT-3 antibody at an initial concentration ofabout 30 ng/mL, and wherein the one or more TNFRSF agonists comprises a4-1BB agonist.

In some embodiments, a combination of IL-2, IL-7, IL-15, and/or IL-21are employed as a combination during the second expansion. In someembodiments, IL-2, IL-7, IL-15, and/or IL-21 as well as any combinationsthereof can be included during the second expansion, including forexample during a Step D processes according to FIG. 18, as well asdescribed herein. In some embodiments, a combination of IL-2, IL-15, andIL-21 are employed as a combination during the second expansion. In someembodiments, IL-2, IL-15, and IL-21 as well as any combinations thereofcan be included during Step D processes according to FIG. 18 and asdescribed herein.

In some embodiments, the second expansion can be conducted in asupplemented cell culture medium comprising IL-2, OKT-3,antigen-presenting feeder cells, and optionally a TNFRSF agonist. Insome embodiments, the second expansion occurs in a supplemented cellculture medium. In some embodiments, the supplemented cell culturemedium comprises IL-2, OKT-3, and antigen-presenting feeder cells. Insome embodiments, the second cell culture medium comprises IL-2, OKT-3,and antigen-presenting cells (APCs; also referred to asantigen-presenting feeder cells). In some embodiments, the secondexpansion occurs in a cell culture medium comprising IL-2, OKT-3, andantigen-presenting feeder cells (i.e., antigen presenting cells).

In some embodiments, the second expansion culture media comprises about500 IU/mL of IL-15, about 400 IU/mL of IL-15, about 300 IU/mL of IL-15,about 200 IU/mL of IL-15, about 180 IU/mL of IL-15, about 160 IU/mL ofIL-15, about 140 IU/mL of IL-15, about 120 IU/mL of IL-15, or about 100IU/mL of IL-15. In some embodiments, the second expansion culture mediacomprises about 500 IU/mL of IL-15 to about 100 IU/mL of IL-15. In someembodiments, the second expansion culture media comprises about 400IU/mL of IL-15 to about 100 IU/mL of IL-15. In some embodiments, thesecond expansion culture media comprises about 300 IU/mL of IL-15 toabout 100 IU/mL of IL-15. In some embodiments, the second expansionculture media comprises about 200 IU/mL of IL-15. In some embodiments,the cell culture medium comprises about 180 IU/mL of IL-15. In anembodiment, the cell culture medium further comprises IL-15. In apreferred embodiment, the cell culture medium comprises about 180 IU/mLof IL-15.

In some embodiments, the second expansion culture media comprises about20 IU/mL of IL-21, about 15 IU/mL of IL-21, about 12 IU/mL of IL-21,about 10 IU/mL of IL-21, about 5 IU/mL of IL-21, about 4 IU/mL of IL-21,about 3 IU/mL of IL-21, about 2 IU/mL of IL-21, about 1 IU/mL of IL-21,or about 0.5 IU/mL of IL-21. In some embodiments, the second expansionculture media comprises about 20 IU/mL of IL-21 to about 0.5 IU/mL ofIL-21. In some embodiments, the second expansion culture media comprisesabout 15 IU/mL of IL-21 to about 0.5 IU/mL of IL-21. In someembodiments, the second expansion culture media comprises about 12 IU/mLof IL-21 to about 0.5 IU/mL of IL-21. In some embodiments, the secondexpansion culture media comprises about 10 IU/mL of IL-21 to about 0.5IU/mL of IL-21. In some embodiments, the second expansion culture mediacomprises about 5 IU/mL of IL-21 to about 1 IU/mL of IL-21. In someembodiments, the second expansion culture media comprises about 2 IU/mLof IL-21. In some embodiments, the cell culture medium comprises about 1IU/mL of IL-21. In some embodiments, the cell culture medium comprisesabout 0.5 IU/mL of IL-21. In an embodiment, the cell culture mediumfurther comprises IL-21. In a preferred embodiment, the cell culturemedium comprises about 1 IU/mL of IL-21.

In some embodiments the antigen-presenting feeder cells (APCs) arePBMCs. In an embodiment, the ratio of TILs to PBMCs and/orantigen-presenting cells in the rapid expansion and/or the secondexpansion is about 1 to 25, about 1 to 50, about 1 to 100, about 1 to125, about 1 to 150, about 1 to 175, about 1 to 200, about 1 to 225,about 1 to 250, about 1 to 275, about 1 to 300, about 1 to 325, about 1to 350, about 1 to 375, about 1 to 400, or about 1 to 500. In anembodiment, the ratio of TILs to PBMCs in the rapid expansion and/or thesecond expansion is between 1 to 50 and 1 to 300. In an embodiment, theratio of TILs to PBMCs in the rapid expansion and/or the secondexpansion is between 1 to 100 and 1 to 200.

In an embodiment, REP and/or the second expansion is performed in flaskswith the bulk TILs being mixed with a 100- or 200-fold excess ofinactivated feeder cells, 30 mg/mL OKT3 anti-CD3 antibody and 3000 IU/mLIL-2 in 150 ml media. Media replacement is done (generally ⅔ mediareplacement via respiration with fresh media) until the cells aretransferred to an alternative growth chamber. Alternative growthchambers include G-REX flasks and gas permeable containers as more fullydiscussed below.

In some embodiments, the second expansion (which can include processesreferred to as the REP process) is shortened to 7-14 days, as discussedin the examples and figures. In some embodiments, the second expansionis shortened to 11 days.

In an embodiment, REP and/or the second expansion may be performed usingT-175 flasks and gas permeable bags as previously described (Tran, etal., J. Immunother. 2008, 31, 742-51; Dudley, et al., J. Immunother.2003, 26, 332-42) or gas permeable cultureware (G-Rex flasks). In someembodiments, the second expansion (including expansions referred to asrapid expansions) is performed in T-175 flasks, and about 1×10⁶ TILssuspended in 150 mL of media may be added to each T-175 flask. The TILsmay be cultured in a 1 to 1 mixture of CM and AIM-V medium, supplementedwith 3000 IU per mL of IL-2 and 30 ng per ml of anti-CD3. The T-175flasks may be incubated at 37° C. in 5% CO₂. Half the media may beexchanged on day 5 using 50/50 medium with 3000 IU per mL of IL-2. Insome embodiments, on day 7 cells from two T-175 flasks may be combinedin a 3 L bag and 300 mL of AIM V with 5% human AB serum and 3000 IU permL of IL-2 was added to the 300 ml of TIL suspension. The number ofcells in each bag was counted every day or two and fresh media was addedto keep the cell count between 0.5 and 2.0×10⁶ cells/mL.

In an embodiment, the second expansion (which can include expansionsreferred to as REP, as well as those referred to in Step D of FIG. 18)may be performed in 500 mL capacity gas permeable flasks with 100 cmgas-permeable silicon bottoms (G-Rex 100, commercially available fromWilson Wolf Manufacturing Corporation, New Brighton, Minn., USA), 5×10⁶or 10×10⁶ TIL may be cultured with PBMCs in 400 mL of 50/50 medium,supplemented with 5% human AB serum, 3000 IU per mL of IL-2 and 30 ngper ml of anti-CD3 (OKT3). The G-Rex 100 flasks may be incubated at 37°C. in 5% CO₂. On day 5, 250 mL of supernatant may be removed and placedinto centrifuge bottles and centrifuged at 1500 rpm (491×g) for 10minutes. The TIL pellets may be re-suspended with 150 mL of fresh mediumwith 5% human AB serum, 3000 IU per mL of IL-2, and added back to theoriginal G-Rex 100 flasks. When TIL are expanded serially in G-Rex 100flasks, on day 7 the TIL in each G-Rex 100 may be suspended in the 300mL of media present in each flask and the cell suspension may be dividedinto 3 100 mL aliquots that may be used to seed 3 G-Rex 100 flasks. Then150 mL of AIM-V with 5% human AB serum and 3000 IU per mL of IL-2 may beadded to each flask. The G-Rex 100 flasks may be incubated at 37° C. in5% CO₂ and after 4 days 150 mL of AIM-V with 3000 IU per mL of IL-2 maybe added to each G-REX 100 flask. The cells may be harvested on day 14of culture.

In an embodiment, the second expansion (including expansions referred toas REP) is performed in flasks with the bulk TILs being mixed with a100- or 200-fold excess of inactivated feeder cells, 30 mg/mL OKT3anti-CD3 antibody and 3000 IU/mL IL-2 in 150 ml media. In someembodiments, media replacement is done until the cells are transferredto an alternative growth chamber. In some embodiments, ⅔ of the media isreplaced by respiration with fresh media. In some embodiments,alternative growth chambers include G-REX flasks and gas permeablecontainers as more fully discussed below.

In an embodiment, the second expansion (including expansions referred toas REP) is performed and further comprises a step wherein TILs areselected for superior tumor reactivity. Any selection method known inthe art may be used. For example, the methods described in U.S. PatentApplication Publication No. 2016/0010058 A1, the disclosures of whichare incorporated herein by reference, may be used for selection of TILsfor superior tumor reactivity.

Optionally, a cell viability assay can be performed after the secondexpansion (including expansions referred to as the REP expansion), usingstandard assays known in the art. For example, a trypan blue exclusionassay can be done on a sample of the bulk TILs, which selectively labelsdead cells and allows a viability assessment. In some embodiments, TILsamples can be counted and viability determined using a Cellometer K2automated cell counter (Nexcelom Bioscience, Lawrence, Mass.).

In some embodiments, the second expansion (including expansions referredto as REP) of TIL can be performed using T-175 flasks and gas-permeablebags as previously described (Tran K Q, Zhou J, Durflinger K H, et al.,2008, J Immunother., 31:742-751, and Dudley M E, Wunderlich J R, SheltonT E, et al. 2003, J Immunother., 26:332-342) or gas-permeable G-Rexflasks. In some embodiments, the second expansion is performed usingflasks. In some embodiments, the second expansion is performed usinggas-permeable G-Rex flasks. In some embodiments, the second expansion isperformed in T-175 flasks, and about 1×10⁶ TIL are suspended in about150 mL of media and this is added to each T-175 flask. The TIL arecultured with irradiated (50 Gy) allogeneic PBMC as “feeder” cells at aratio of 1 to 100 and the cells were cultured in a 1 to 1 mixture of CMand AIM-V medium (50/50 medium), supplemented with 3000 IU/mL of IL-2and 30 ng/mL of anti-CD3. The T-175 flasks are incubated at 37° C. in 5%CO₂. In some embodiments, half the media is changed on day 5 using 50/50medium with 3000 IU/mL of IL-2. In some embodiments, on day 7, cellsfrom 2 T-175 flasks are combined in a 3 L bag and 300 mL of AIM-V with5% human AB serum and 3000 IU/mL of IL-2 is added to the 300 mL of TILsuspension. The number of cells in each bag can be counted every day ortwo and fresh media can be added to keep the cell count between about0.5 and about 2.0×10⁶ cells/mL.

In some embodiments, the second expansion (including expansions referredto as REP) are performed in 500 mL capacity flasks with 100 cm²gas-permeable silicon bottoms (G-Rex 100, Wilson Wolf) (FIG. 1), about5×10⁶ or 10×10⁶ TIL are cultured with irradiated allogeneic PBMC at aratio of 1 to 100 in 400 mL of 50/50 medium, supplemented with 3000IU/mL of IL-2 and 30 ng/mL of anti-CD3. The G-Rex 100 flasks areincubated at 37° C. in 5% CO₂. In some embodiments, on day 5, 250 mL ofsupernatant is removed and placed into centrifuge bottles andcentrifuged at 1500 rpm (491 g) for 10 minutes. The TIL pellets can thenbe resuspended with 150 mL of fresh 50/50 medium with 3000 IU/mL of IL-2and added back to the original G-Rex 100 flasks. In embodiments whereTILs are expanded serially in G-Rex 100 flasks, on day 7 the TIL in eachG-Rex 100 are suspended in the 300 mL of media present in each flask andthe cell suspension was divided into three 100 mL aliquots that are usedto seed 3 G-Rex 100 flasks. Then 150 mL of AIM-V with 5% human AB serumand 3000 IU/mL of IL-2 is added to each flask. The G-Rex 100 flasks areincubated at 37° C. in 5% CO₂ and after 4 days 150 mL of AIM-V with 3000IU/mL of IL-2 is added to each G-Rex 100 flask. The cells are harvestedon day 14 of culture.

The diverse antigen receptors of T and B lymphocytes are produced bysomatic recombination of a limited, but large number of gene segments.These gene segments: V (variable), D (diversity), J (joining), and C(constant), determine the binding specificity and downstreamapplications of immunoglobulins and T-cell receptors (TCRs). The presentinvention provides a method for generating TILs which exhibit andincrease the T-cell repertoire diversity. In some embodiments, the TILsobtained by the present method exhibit an increase in the T-cellrepertoire diversity. In some embodiments, the TILs obtained in thesecond expansion exhibit an increase in the T-cell repertoire diversity.In some embodiments, the increase in diversity is an increase in theimmunoglobulin diversity and/or the T-cell receptor diversity. In someembodiments, the diversity is in the immunoglobulin is in theimmunoglobulin heavy chain. In some embodiments, the diversity is in theimmunoglobulin is in the immunoglobulin light chain. In someembodiments, the diversity is in the T-cell receptor. In someembodiments, the diversity is in one of the T-cell receptors selectedfrom the group consisting of alpha, beta, gamma, and delta receptors. Insome embodiments, there is an increase in the expression of T-cellreceptor (TCR) alpha and/or beta. In some embodiments, there is anincrease in the expression of T-cell receptor (TCR) alpha. In someembodiments, there is an increase in the expression of T-cell receptor(TCR) beta. In some embodiments, there is an increase in the expressionof TCRab (i.e., TCRα/β).

In some embodiments, the second expansion culture medium (e.g.,sometimes referred to as CM2 or the second cell culture medium),comprises IL-2, OKT-3, as well as the antigen-presenting feeder cells(APCs), as discussed in more detail below.

In some embodiments, the second expansion, for example, Step D accordingto FIG. 18, is performed in a closed system bioreactor. In someembodiments, a closed system is employed for the TIL expansion, asdescribed herein. In some embodiments, a single bioreactor is employed.In some embodiments, the single bioreactor employed is for example aG-REX-10 or a G-REX-100. In some embodiments, the closed systembioreactor is a single bioreactor.

Feeder Cells and Antigen Presenting Cells

In an embodiment, the second expansion procedures described herein (forexample including expansion such as those described in Step D from FIG.18, as well as those referred to as REP) require an excess of feedercells during REP TIL expansion and/or during the second expansion. Inmany embodiments, the feeder cells are peripheral blood mononuclearcells (PBMCs) obtained from standard whole blood units from healthyblood donors. The PBMCs are obtained using standard methods such asFicoll-Paque gradient separation.

In general, the allogenic PBMCs are inactivated, either via irradiationor heat treatment, and used in the REP procedures, as described in theexamples, which provides an exemplary protocol for evaluating thereplication incompetence of irradiate allogeneic PBMCs.

In some embodiments, PBMCs are considered replication incompetent andaccepted for use in the TIL expansion procedures described herein if thetotal number of viable cells on day 14 is less than the initial viablecell number put into culture on day 0 of the REP and/or day 0 of thesecond expansion (i.e., the start day of the second expansion).

In some embodiments, PBMCs are considered replication incompetent andaccepted for use in the TIL expansion procedures described herein if thetotal number of viable cells, cultured in the presence of OKT3 and IL-2,on day 7 and day 14 has not increased from the initial viable cellnumber put into culture on day 0 of the REP and/or day 0 of the secondexpansion (i.e., the start day of the second expansion). In someembodiments, the PBMCs are cultured in the presence of 30 ng/ml OKT3antibody and 3000 IU/ml IL-2.

In some embodiments, PBMCs are considered replication incompetent andaccepted for use in the TIL expansion procedures described herein if thetotal number of viable cells, cultured in the presence of OKT3 and IL-2,on day 7 and day 14 has not increased from the initial viable cellnumber put into culture on day 0 of the REP and/or day 0 of the secondexpansion (i.e., the start day of the second expansion). In someembodiments, the PBMCs are cultured in the presence of 5-60 ng/ml OKT3antibody and 1000-6000 IU/ml IL-2. In some embodiments, the PBMCs arecultured in the presence of 10-50 ng/ml OKT3 antibody and 2000-5000IU/ml IL-2. In some embodiments, the PBMCs are cultured in the presenceof 20-40 ng/ml OKT3 antibody and 2000-4000 IU/ml IL-2. In someembodiments, the PBMCs are cultured in the presence of 25-35 ng/ml OKT3antibody and 2500-3500 IU/ml IL-2.

In some embodiments, the antigen-presenting feeder cells are PBMCs. Insome embodiments, the antigen-presenting feeder cells are artificialantigen-presenting feeder cells. In an embodiment, the ratio of TILs toantigen-presenting feeder cells in the second expansion is about 1 to25, about 1 to 50, about 1 to 100, about 1 to 125, about 1 to 150, about1 to 175, about 1 to 200, about 1 to 225, about 1 to 250, about 1 to275, about 1 to 300, about 1 to 325, about 1 to 350, about 1 to 375,about 1 to 400, or about 1 to 500. In an embodiment, the ratio of TILsto antigen-presenting feeder cells in the second expansion is between 1to 50 and 1 to 300. In an embodiment, the ratio of TILs toantigen-presenting feeder cells in the second expansion is between 1 to100 and 1 to 200.

In an embodiment, the second expansion procedures described hereinrequire a ratio of about 2.5×10⁹ feeder cells to about 100×10⁶ TILs. Inanother embodiment, the second expansion procedures described hereinrequire a ratio of about 2.5×10⁹ feeder cells to about 50×10⁶ TILs. Inyet another embodiment, the second expansion procedures described hereinrequire about 2.5×10⁹ feeder cells to about 25×10⁶ TILs.

In an embodiment, the second expansion procedures described hereinrequire an excess of feeder cells during the second expansion. In manyembodiments, the feeder cells are peripheral blood mononuclear cells(PBMCs) obtained from standard whole blood units from healthy blooddonors. The PBMCs are obtained using standard methods such asFicoll-Paque gradient separation. In an embodiment, artificialantigen-presenting (aAPC) cells are used in place of PBMCs.

In general, the allogenic PBMCs are inactivated, either via irradiationor heat treatment, and used in the TIL expansion procedures describedherein, including the exemplary procedures described in the figures andexamples.

In an embodiment, artificial antigen presenting cells are used in thesecond expansion as a replacement for, or in combination with, PBMCs.

Cytokines

The expansion methods described herein generally use culture media withhigh doses of a cytokine, in particular IL-2, as is known in the art.

Alternatively, using combinations of cytokines for the rapid expansionand or second expansion of TILS is additionally possible, withcombinations of two or more of IL-2, IL-15 and IL-21 as is generallyoutlined in International Publication No. WO 2015/189356 and WInternational Publication No. WO 2015/189357, hereby expresslyincorporated by reference in their entirety. Thus, possible combinationsinclude IL-2 and IL-15, IL-2 and IL-21, IL-15 and IL-21 and IL-2, IL-15and IL-21, with the latter finding particular use in many embodiments.The use of combinations of cytokines specifically favors the generationof lymphocytes, and in particular T-cells as described therein.

Step E: Harvest TILS

After the second expansion step, cells can be harvested. In someembodiments the TILs are harvested after one, two, three, four or moreexpansion steps, for example as provided in FIG. 18. In some embodimentsthe TILs are harvested after two expansion steps, for example asprovided in FIG. 18.

TILs can be harvested in any appropriate and sterile manner, includingfor example by centrifugation. Methods for TIL harvesting are well knownin the art and any such know methods can be employed with the presentprocess. In some embodiments, TILS are harvest using an automatedsystem.

Cell harvesters and/or cell processing systems are commerciallyavailable from a variety of sources, including, for example, FreseniusKabi, Tomtec Life Science, Perkin Elmer, and Inotech BiosystemsInternational, Inc. Any cell based harvester can be employed with thepresent methods. In some embodiments, the cell harvester and/or cellprocessing systems is a membrane-based cell harvester. In someembodiments, cell harvesting is via a cell processing system, such asthe LOVO system (manufactured by Fresenius Kabi). The term “LOVO cellprocessing system” also refers to any instrument or device manufacturedby any vendor that can pump a solution comprising cells through amembrane or filter such as a spinning membrane or spinning filter in asterile and/or closed system environment, allowing for continuous flowand cell processing to remove supernatant or cell culture media withoutpelletization. In some embodiments, the cell harvester and/or cellprocessing system can perform cell separation, washing, fluid-exchange,concentration, and/or other cell processing steps in a closed, sterilesystem.

In some embodiments, the harvest, for example, Step E according to FIG.18, is performed from a closed system bioreactor. In some embodiments, aclosed system is employed for the TIL expansion, as described herein. Insome embodiments, a single bioreactor is employed. In some embodiments,the single bioreactor employed is for example a G-REX-10 or a G-REX-100.In some embodiments, the closed system bioreactor is a singlebioreactor.

In some embodiments, Step E according to FIG. 18, is performed accordingto the processes described in Example 7. In some embodiments, the closedsystem is accessed via syringes under sterile conditions in order tomaintain the sterility and closed nature of the system. In someembodiments, a closed system as described in Example 7 is employed.

In some embodiments, TILs are harvested according to the methodsdescribed in the Examples.

Step F: Final Formulation/Transfer to Infusion Bag

After Steps A through E as provided in an exemplary order in FIG. 18 andas outlined in detailed above and herein are complete, cells aretransferred to a container for use in administration to a patient. Insome embodiments, once a therapeutically sufficient number of TILs areobtained using the expansion methods described above, they aretransferred to a container for use in administration to a patient.

In an embodiment, TILs expanded using APCs of the present disclosure areadministered to a patient as a pharmaceutical composition. In anembodiment, the pharmaceutical composition is a suspension of TILs in asterile buffer. TILs expanded using PBMCs of the present disclosure maybe administered by any suitable route as known in the art. In someembodiments, the T-cells are administered as a single intra-arterial orintravenous infusion, which preferably lasts approximately 30 to 60minutes. Other suitable routes of administration includeintraperitoneal, intrathecal, and intralymphatic.

Optional Cell Medium Components

1. Anti-CD3 Antibodies

In some embodiments, the culture media used in expansion methodsdescribed herein (including those referred to as REP, see for example,FIG. 18) also includes an anti-CD3 antibody. An anti-CD3 antibody incombination with IL-2 induces T cell activation and cell division in theTIL population. This effect can be seen with full length antibodies aswell as Fab and F(ab′)2 fragments, with the former being generallypreferred; see, e.g., Tsoukas et al., J. Immunol. 1985, 135, 1719,hereby incorporated by reference in its entirety.

As will be appreciated by those in the art, there are a number ofsuitable anti-human CD3 antibodies that find use in the invention,including anti-human CD3 polyclonal and monoclonal antibodies fromvarious mammals, including, but not limited to, murine, human, primate,rat, and canine antibodies. In particular embodiments, the OKT3 anti-CD3antibody is used (commercially available from Ortho-McNeil, Raritan,N.J. or Miltenyi Biotech, Auburn, Calif.).

TABLE 5 Amino acid sequences of muromonab (exemplary OKT-3 antibody)Identifier Sequence (One-Letter Amino Acid Symbols) SEQ ID NO: 1QVQLQQSGAE LARPGASVEM SCKASGYTFT RYTMHWVFQR PGQGLEWIGY INPSRGYTNY 60Muromonab heavy NQKFKDKATL TTDKSSSTAY MQLSSLTSED SAVYYCARYY DDHYCLDYWGQGTTLTVSSA 120 chain KTTAPSVYPL APVCGGTTGS SVTLGCLVEG YFPEPVTLTWNSGSLSSGVH TFPAVLQSDL 180 YTLSSSVTVT SSTWPSQSIT CNVAHPASST KVDKKIEPRPKSCDKTHTCP PCPAPELLGG 240 PSVFLEPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNWYVDGVEVHNA KTKPREEQYN 300 STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTISKAKGQPREPQ VYTLPPSRDE 360 LTENQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPVLDSDGSFFLY SKLTVDESRW 420 QQGNVFSCSV MHEALHNHYT QKSLSLSPGK 450 SEQ IDNO: 2 QIVLTQSPAI MSASPGEKVT MTCSASSSVS YMNWYQQKSG TSPKRWIYDT SKLASGVPAH60 Muromonab light FRGSGSGTSY SLTISGMEAE DAATYYCQQW SSNPFTFGSGTKLEINRADT APTVSIFPPS 120 chain SEQLTSGGAS VVCFLNNFYP KDINVKWKIDGSERQNGVLN SWTDQDSKDS TYSMSSTLTL 180 TKDEYERHNS YTCEATHKTS TSPIVKSFNRNEC 213

2. 4-1BB (CD137) AGONISTS

In an embodiment, the TNFRSF agonist is a 4-1BB (CD137) agonist. The4-1BB agonist may be any 4-1BB binding molecule known in the art. The4-1BB binding molecule may be a monoclonal antibody or fusion proteincapable of binding to human or mammalian 4-1BB. The 4-1BB agonists or4-1BB binding molecules may comprise an immunoglobulin heavy chain ofany isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1,IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.The 4-1BB agonist or 4-1BB binding molecule may have both a heavy and alight chain. As used herein, the term binding molecule also includesantibodies (including full length antibodies), monoclonal antibodies(including full length monoclonal antibodies), polyclonal antibodies,multispecific antibodies (e.g., bispecific antibodies), human, humanizedor chimeric antibodies, and antibody fragments, e.g., Fab fragments,F(ab′) fragments, fragments produced by a Fab expression library,epitope-binding fragments of any of the above, and engineered forms ofantibodies, e.g., scFv molecules, that bind to 4-1BB. In an embodiment,the 4-1BB agonist is an antigen binding protein that is a fully humanantibody. In an embodiment, the 4-1BB agonist is an antigen bindingprotein that is a humanized antibody. In some embodiments, 4-1BBagonists for use in the presently disclosed methods and compositionsinclude anti-4-1BB antibodies, human anti-4-1BB antibodies, mouseanti-4-1BB antibodies, mammalian anti-4-1BB antibodies, monoclonalanti-4-1BB antibodies, polyclonal anti-4-1BB antibodies, chimericanti-4-1BB antibodies, anti-4-1BB adnectins, anti-4-1BB domainantibodies, single chain anti-4-1BB fragments, heavy chain anti-4-1BBfragments, light chain anti-4-1BB fragments, anti-4-1BB fusion proteins,and fragments, derivatives, conjugates, variants, or biosimilarsthereof. Agonistic anti-4-1BB antibodies are known to induce strongimmune responses. Lee, et al., PLOS One 2013, 8, e69677. In a preferredembodiment, the 4-1BB agonist is an agonistic, anti-4-1BB humanized orfully human monoclonal antibody (i.e., an antibody derived from a singlecell line). In an embodiment, the 4-1BB agonist is EU-101 (Eutilex Co.Ltd.), utomilumab, or urelumab, or a fragment, derivative, conjugate,variant, or biosimilar thereof. In a preferred embodiment, the 4-1BBagonist is utomilumab or urelumab, or a fragment, derivative, conjugate,variant, or biosimilar thereof.

In a preferred embodiment, the 4-1BB agonist or 4-1BB binding moleculemay also be a fusion protein. In a preferred embodiment, a multimeric4-1BB agonist, such as a trimeric or hexameric 4-1BB agonist (with threeor six ligand binding domains), may induce superior receptor (4-1BBL)clustering and internal cellular signaling complex formation compared toan agonistic monoclonal antibody, which typically possesses two ligandbinding domains. Trimeric (trivalent) or hexameric (or hexavalent) orgreater fusion proteins comprising three TNFRSF binding domains andIgG1-Fc and optionally further linking two or more of these fusionproteins are described, e.g., in Gieffers, et al., Mol. CancerTherapeutics 2013, 12, 2735-47.

Agonistic 4-1BB antibodies and fusion proteins are known to inducestrong immune responses. In a preferred embodiment, the 4-1BB agonist isa monoclonal antibody or fusion protein that binds specifically to 4-1BBantigen in a manner sufficient to reduce toxicity. In some embodiments,the 4-1BB agonist is an agonistic 4-1BB monoclonal antibody or fusionprotein that abrogates antibody-dependent cellular toxicity (ADCC), forexample NK cell cytotoxicity. In some embodiments, the 4-1BB agonist isan agonistic 4-1BB monoclonal antibody or fusion protein that abrogatesantibody-dependent cell phagocytosis (ADCP). In some embodiments, the4-1BB agonist is an agonistic 4-1BB monoclonal antibody or fusionprotein that abrogates complement-dependent cytotoxicity (CDC). In someembodiments, the 4-1BB agonist is an agonistic 4-1BB monoclonal antibodyor fusion protein which abrogates Fc region functionality.

In some embodiments, the 4-1BB agonists are characterized by binding tohuman 4-1BB (SEQ ID NO:9) with high affinity and agonistic activity. Inan embodiment, the 4-1BB agonist is a binding molecule that binds tohuman 4-1BB (SEQ ID NO:9). In an embodiment, the 4-1BB agonist is abinding molecule that binds to murine 4-1BB (SEQ ID NO:10). The aminoacid sequences of 4-1BB antigen to which a 4-1BB agonist or bindingmolecule binds are summarized in TABLE 6.

TABLE 6 Amino acid sequences of 4-1BB antigens. Identifier Sequence(One-Letter Amino Acid Symbols) SEQ ID NO: 9 MGNSCYNIVA TLLLVLNFERTRSLQDPCSN CPAGTFCDNN RNQICSPCPP NSFSSAGGQR 60 human 4-1BB, TCDICRQCKGVFRTRKECSS TSNAECDCTP GFHCLGAGCS MCEQDCKQGQ ELTKKGCKDC 120 Tumornecrosis CFGTFNDQKR GICRPWTNCS LDGKSVLVNG TKERDVVCGP SPADLSPGASSVTPPAPARE 180 factor receptor PGHSPQIISF FLALTSTALL FLLFFLTLRFSVVKRGRKKL LYIFKQPFMR PVQTTQEEDG 240 superfamily, CSCRFPEEEE GGCEL 255member 9 (Homo sapiens) SEQ ID NO: 10 MGNNCYNVVV IVLLLVGCEK VGAVQNSCDNCQPGTFCRKY NPVCKSCPPS TFSSIGGQPN 60 murine 4-1BB, CNICRVCAGY FRFKKFCSSTHNAECECIEG FHCLGPQCTR CEKDCRPGQE LTKQGCKTCS 120 Tumor necrosisLGTFNDQNGT GVCRPWTNCS LDGRSVLKTG TTEKDVVCGP PVVSFSPSTT ISVTPEGGPG 180factor receptor GHSLQVLTLF LALTSALLLA LIFITLLFSV LKWIRKKFPH IFKQPFKKTTGAAQEEDACS 240 superfamily, CRCPQEEEGG GGGYEL 256 member 9 (Musmusculus)

In some embodiments, the compositions, processes and methods describedinclude a 4-1BB agonist that binds human or murine 4-1BB with a K_(D) ofabout 100 pM or lower, binds human or murine 4-1BB with a K_(D) of about90 pM or lower, binds human or murine 4-1BB with a K_(D) of about 80 pMor lower, binds human or murine 4-1BB with a K_(D) of about 70 pM orlower, binds human or murine 4-1BB with a K_(D) of about 60 pM or lower,binds human or murine 4-1BB with a K_(D) of about 50 pM or lower, bindshuman or murine 4-1BB with a K_(D) of about 40 pM or lower, or bindshuman or murine 4-1BB with a K_(D) of about 30 pM or lower.

In some embodiments, the compositions, processes and methods describedinclude a 4-1BB agonist that binds to human or murine 4-1BB with ak_(assoc) of about 7.5×10⁵ 1/M·s or faster, binds to human or murine4-1BB with a k_(assoc) of about 7.5×10⁵ 1/M·s or faster, binds to humanor murine 4-1BB with a k_(assoc) of about 8×10⁵ 1/M·s or faster, bindsto human or murine 4-1BB with a k_(assoc) of about 8.5×10⁵ 1/M·s orfaster, binds to human or murine 4-1BB with a k_(assoc) of about 9×10⁵1/M·s or faster, binds to human or murine 4-1BB with a k_(assoc) ofabout 9.5×10⁵ 1/M·s or faster, or binds to human or murine 4-1BB with ak_(assoc) of about 1×10⁶ 1/M·s or faster.

In some embodiments, the compositions, processes and methods describedinclude a 4-1BB agonist that binds to human or murine 4-1BB with ak_(dissoc) of about 2×10⁻⁵ 1/s or slower, binds to human or murine 4-1BBwith a k_(dissoc) of about 2.1×10⁻⁵ 1/s or slower, binds to human ormurine 4-1BB with a k_(dissoc) of about 2.2×10⁻⁵ 1/s or slower, binds tohuman or murine 4-1BB with a k_(dissoc) of about 2.3×10⁻⁵ 1/s or slower,binds to human or murine 4-1BB with a k_(dissoc) of about 2.4×10⁻⁵ 1/sor slower, binds to human or murine 4-1BB with a k_(dissoc) of about2.5×10⁻⁵ 1/s or slower, binds to human or murine 4-1BB with a k_(dissoc)of about 2.6×10⁻⁵ 1/s or slower or binds to human or murine 4-1BB with ak_(dissoc) of about 2.7×10⁻⁵ 1/s or slower, binds to human or murine4-1BB with a k_(dissoc) of about 2.8×10⁻⁵ 1/s or slower, binds to humanor murine 4-1BB with a k_(dissoc) of about 2.9×10⁻⁵ 1/s or slower, orbinds to human or murine 4-1BB with a k_(dissoc) of about 3×10⁻⁵ 1/s orslower.

In some embodiments, the compositions, processes and methods describedinclude a 4-1BB agonist that binds to human or murine 4-1BB with an IC₅₀of about 10 nM or lower, binds to human or murine 4-1BB with an IC₅₀ ofabout 9 nM or lower, binds to human or murine 4-1BB with an IC₅₀ ofabout 8 nM or lower, binds to human or murine 4-1BB with an IC₅₀ ofabout 7 nM or lower, binds to human or murine 4-1BB with an IC₅₀ ofabout 6 nM or lower, binds to human or murine 4-1BB with an IC₅₀ ofabout 5 nM or lower, binds to human or murine 4-1BB with an IC₅₀ ofabout 4 nM or lower, binds to human or murine 4-1BB with an IC₅₀ ofabout 3 nM or lower, binds to human or murine 4-1BB with an IC₅₀ ofabout 2 nM or lower, or binds to human or murine 4-1BB with an IC₅₀ ofabout 1 nM or lower.

In a preferred embodiment, the 4-1BB agonist is utomilumab, also knownas PF-05082566 or MOR-7480, or a fragment, derivative, variant, orbiosimilar thereof. Utomilumab is available from Pfizer, Inc. Utomilumabis an immunoglobulin G2-lambda, anti-[Homo sapiens TNFRSF9 (tumornecrosis factor receptor (TNFR) superfamily member 9, 4-1BB, T cellantigen ILA, CD137)], Homo sapiens (fully human) monoclonal antibody.The amino acid sequences of utomilumab are set forth in Table 7.Utomilumab comprises glycosylation sites at Asn59 and Asn292; heavychain intrachain disulfide bridges at positions 22-96 (V_(H)-V_(L)),143-199 (C_(H)1-C_(L)), 256-316 (C_(H)2) and 362-420 (C_(H)3); lightchain intrachain disulfide bridges at positions 22′-87′ (V_(H)-V_(L))and 136′-195′ (C_(H)1-C_(L)); interchain heavy chain-heavy chaindisulfide bridges at IgG2A isoform positions 218-218, 219-219, 222-222,and 225-225, at IgG2A/B isoform positions 218-130, 219-219, 222-222, and225-225, and at IgG2B isoform positions 219-130 (2), 222-222, and225-225; and interchain heavy chain-light chain disulfide bridges atIgG2A isoform positions 130-213′ (2), IgG2A/B isoform positions 218-213′and 130-213′, and at IgG2B isoform positions 218-213′ (2). Thepreparation and properties of utomilumab and its variants and fragmentsare described in U.S. Pat. Nos. 8,821,867; 8,337,850; and 9,468,678, andInternational Patent Application Publication No. WO 2012/032433 A1, thedisclosures of each of which are incorporated by reference herein.Preclinical characteristics of utomilumab are described in Fisher, etal., Cancer Immunolog. & Immunother. 2012, 61, 1721-33. Current clinicaltrials of utomilumab in a variety of hematological and solid tumorindications include U.S. National Institutes of Healthclinicaltrials.gov identifiers NCT02444793, NCT01307267, NCT02315066,and NCT02554812.

In an embodiment, a 4-1BB agonist comprises a heavy chain given by SEQID NO:11 and a light chain given by SEQ ID NO:12. In an embodiment, a4-1BB agonist comprises heavy and light chains having the sequencesshown in SEQ ID NO:11 and SEQ ID NO:12, respectively, or antigen bindingfragments, Fab fragments, single-chain variable fragments (scFv),variants, or conjugates thereof. In an embodiment, a 4-1BB agonistcomprises heavy and light chains that are each at least 99% identical tothe sequences shown in SEQ ID NO:11 and SEQ ID NO:12, respectively. Inan embodiment, a 4-1BB agonist comprises heavy and light chains that areeach at least 98% identical to the sequences shown in SEQ ID NO:11 andSEQ ID NO:12, respectively. In an embodiment, a 4-1BB agonist comprisesheavy and light chains that are each at least 97% identical to thesequences shown in SEQ ID NO:11 and SEQ ID NO:12, respectively. In anembodiment, a 4-1BB agonist comprises heavy and light chains that areeach at least 96% identical to the sequences shown in SEQ ID NO:11 andSEQ ID NO:12, respectively. In an embodiment, a 4-1BB agonist comprisesheavy and light chains that are each at least 95% identical to thesequences shown in SEQ ID NO:11 and SEQ ID NO:12, respectively.

In an embodiment, the 4-1BB agonist comprises the heavy and light chainCDRs or variable regions (VRs) of utomilumab. In an embodiment, the4-1BB agonist heavy chain variable region (V_(H)) comprises the sequenceshown in SEQ ID NO:13, and the 4-1BB agonist light chain variable region(V_(L)) comprises the sequence shown in SEQ ID NO:14, and conservativeamino acid substitutions thereof. In an embodiment, a 4-1BB agonistcomprises V_(H) and V_(L) regions that are each at least 99% identicalto the sequences shown in SEQ ID NO:13 and SEQ ID NO:14, respectively.In an embodiment, a 4-1BB agonist comprises V_(H) and V_(L) regions thatare each at least 98% identical to the sequences shown in SEQ ID NO:13and SEQ ID NO:14, respectively. In an embodiment, a 4-1BB agonistcomprises V_(H) and V_(L) regions that are each at least 97% identicalto the sequences shown in SEQ ID NO:13 and SEQ ID NO:14, respectively.In an embodiment, a 4-1BB agonist comprises V_(H) and V_(L) regions thatare each at least 96% identical to the sequences shown in SEQ ID NO:13and SEQ ID NO:14, respectively. In an embodiment, a 4-1BB agonistcomprises V_(H) and V_(L) regions that are each at least 95% identicalto the sequences shown in SEQ ID NO:13 and SEQ ID NO:14, respectively.In an embodiment, a 4-1BB agonist comprises an scFv antibody comprisingV_(H) and V_(L) regions that are each at least 99% identical to thesequences shown in SEQ ID NO:13 and SEQ ID NO:14.

In an embodiment, a 4-1BB agonist comprises heavy chain CDR1, CDR2 andCDR3 domains having the sequences set forth in SEQ ID NO:15, SEQ IDNO:16, and SEQ ID NO:17, respectively, and conservative amino acidsubstitutions thereof, and light chain CDR1, CDR2 and CDR3 domainshaving the sequences set forth in SEQ ID NO:18, SEQ ID NO:19, and SEQ IDNO:20, respectively, and conservative amino acid substitutions thereof.

In an embodiment, the 4-1BB agonist is a 4-1BB agonist biosimilarmonoclonal antibody approved by drug regulatory authorities withreference to utomilumab. In an embodiment, the biosimilar monoclonalantibody comprises an 4-1BB antibody comprising an amino acid sequencewhich has at least 97% sequence identity, e.g., 97%, 98%, 99% or 100%sequence identity, to the amino acid sequence of a reference medicinalproduct or reference biological product and which comprises one or morepost-translational modifications as compared to the reference medicinalproduct or reference biological product, wherein the reference medicinalproduct or reference biological product is utomilumab. In someembodiments, the one or more post-translational modifications areselected from one or more of: glycosylation, oxidation, deamidation, andtruncation. In some embodiments, the biosimilar is a 4-1BB agonistantibody authorized or submitted for authorization, wherein the 4-1BBagonist antibody is provided in a formulation which differs from theformulations of a reference medicinal product or reference biologicalproduct, wherein the reference medicinal product or reference biologicalproduct is utomilumab. The 4-1BB agonist antibody may be authorized by adrug regulatory authority such as the U.S. FDA and/or the EuropeanUnion's EMA. In some embodiments, the biosimilar is provided as acomposition which further comprises one or more excipients, wherein theone or more excipients are the same or different to the excipientscomprised in a reference medicinal product or reference biologicalproduct, wherein the reference medicinal product or reference biologicalproduct is utomilumab. In some embodiments, the biosimilar is providedas a composition which further comprises one or more excipients, whereinthe one or more excipients are the same or different to the excipientscomprised in a reference medicinal product or reference biologicalproduct, wherein the reference medicinal product or reference biologicalproduct is utomilumab.

TABLE 7 Amino acid sequences for 4-1BB agonist antibodies related toutomilumab. Identifier Sequence (One-Letter Amino Acid Symbols) SEQ IDNO: 11 EVQLVQSGAE VKKPGESLRI SCKGSGYSFS TYWISWVRQM PGKGLEWMGK IYPGDSYTNY60 heavy chain for SPSFQGQVTI SADKSISTAY LQWSSLKASD TAMYYCARGYGIFDYWGQGT LVTVSSASTK 120 utomilumab GPSVFPLAPC SRSTSESTAA LGCLVKDYFPEPVTVSWNSG ALTSGVHTFP AVLQSSGLYS 180 LSSVVTVPSS NFGTQTYTCN VDHKPSNTKVDKTVERKCCV ECPPCPAPPV AGPSVFLFPP 240 KPKDTLMISR TPEVTCVVVD VSHEDPEVQFNWYVDGVEVH NAKTKPREEQ FNSTFRVVSV 300 LTVVHQDWLN GKEYKCKVSN KGLPAPIEKTISKTKGQPRE PQVYTLPPSR EEMTKNQVSL 360 TCLVKGFYPS DIAVEWESNG QPENNYKTTPPMLDSDGSFF LYSKLTVDKS RWQQGNVFSC 420 SVMHEALHNH YTQKSLSLSP G 441 SEQ IDNO: 12 SYELTQPPSV SVSPGQTASI TCSGDNIGDQ YAHWYQQKPG QSPVLVIYQD KNRPSGIPER60 light chain for FSGSNSGNTA TLTISGTQAM DEADYYCATY TGFGSLAVFGGGTKLTVLGQ PKAAPSVTLF 120 utomilumab PPSSEELQAN KATLVCLISD FYPGAVTVAWKADSSPVKAG VETTTPSKQS NNKYAASSYL 180 SLTPEQWKSH RSYSCQVTHE GSTVEKTVAPTECS 214 SEQ ID NO: 13 EVQLVQSGAE VKKPGESLRI SCKGSGYSFS TYWISWVRQMPGKGLEWMG KIYPGDSYTN 60 heavy chain YSPSFQGQVT ISADKSISTA YLQWSSLKASDTAMYYCARG YGIFDYWGQ GTLVTVSS 118 variable region for utomilumab SEQ IDNO: 14 SYELTQPPSV SVSPGQTASI TCSGDNIGDQ YAHWYQQKPG QSPVLVIYQD KNRPSGIPER60 light chain FSGSNSGNTA TLTISGTQAM DEADYYCATY TGFGSLAVFG GGTKLTVL 108variable region for utomilumab SEQ ID NO: 15 STYWIS 6 heavy chain CDR1for utomilumab SEQ ID NO: 16 KIYPGDSYTN YSPSFQG 17 heavy chain CDR2 forutomilumab SEQ ID NO: 17 RGYGIFDY 8 heavy chain CDR3 for utomilumab SEQID NO: 18 SGDNIGDQYA H 11 light chain CDR1 for utomilumab SEQ ID NO: 19QDKNRPS 7 light chain CDR2 for utomilumab SEQ ID NO: 20 ATYTGFGSLA V 11light chain CDR3 for utomilumab

In a preferred embodiment, the 4-1BB agonist is the monoclonal antibodyurelumab, also known as BMS-663513 and 20H4.9.h4a, or a fragment,derivative, variant, or biosimilar thereof. Urelumab is available fromBristol-Myers Squibb, Inc., and Creative Biolabs, Inc. Urelumab is animmunoglobulin G4-kappa, anti-[Homo sapiens TNFRSF9 (tumor necrosisfactor receptor superfamily member 9, 4-1BB, T cell antigen ILA,CD137)], Homo sapiens (fully human) monoclonal antibody. The amino acidsequences of urelumab are set forth in Table 7. Urelumab comprisesN-glycosylation sites at positions 298 (and 298″); heavy chainintrachain disulfide bridges at positions 22-95 (V_(H)-V_(L)), 148-204(C_(H)1-C_(L)), 262-322 (C_(H)2) and 368-426 (C_(H)3) (and at positions22″-95″, 148″-204″, 262″-322″, and 368″-426″); light chain intrachaindisulfide bridges at positions 23′-88′ (V_(H)-V_(L)) and 136′-196′(C_(H)1-C_(L)) (and at positions 23′-88′ and 136″′-196″′); interchainheavy chain-heavy chain disulfide bridges at positions 227-227″ and230-230″; and interchain heavy chain-light chain disulfide bridges at135-216′ and 135″-216′″. The preparation and properties of urelumab andits variants and fragments are described in U.S. Pat. Nos. 7,288,638 and8,962,804, the disclosures of which are incorporated by referenceherein. The preclinical and clinical characteristics of urelumab aredescribed in Segal, et al., Clin. Cancer Res. 2016, available athttp:/dx.doi.org/10.1158/1078-0432.CCR-16-1272. Current clinical trialsof urelumab in a variety of hematological and solid tumor indicationsinclude U.S. National Institutes of Health clinicaltrials.govidentifiers NCT01775631, NCT02110082, NCT02253992, and NCT01471210.

In an embodiment, a 4-1BB agonist comprises a heavy chain given by SEQID NO:21 and a light chain given by SEQ ID NO:22. In an embodiment, a4-1BB agonist comprises heavy and light chains having the sequencesshown in SEQ ID NO:21 and SEQ ID NO:22, respectively, or antigen bindingfragments, Fab fragments, single-chain variable fragments (scFv),variants, or conjugates thereof. In an embodiment, a 4-1BB agonistcomprises heavy and light chains that are each at least 99% identical tothe sequences shown in SEQ ID NO:21 and SEQ ID NO:22, respectively. Inan embodiment, a 4-1BB agonist comprises heavy and light chains that areeach at least 98% identical to the sequences shown in SEQ ID NO:21 andSEQ ID NO:22, respectively. In an embodiment, a 4-1BB agonist comprisesheavy and light chains that are each at least 97% identical to thesequences shown in SEQ ID NO:21 and SEQ ID NO:22, respectively. In anembodiment, a 4-1BB agonist comprises heavy and light chains that areeach at least 96% identical to the sequences shown in SEQ ID NO:21 andSEQ ID NO:22, respectively. In an embodiment, a 4-1BB agonist comprisesheavy and light chains that are each at least 95% identical to thesequences shown in SEQ ID NO:21 and SEQ ID NO:22, respectively.

In an embodiment, the 4-1BB agonist comprises the heavy and light chainCDRs or variable regions (VRs) of urelumab. In an embodiment, the 4-1BBagonist heavy chain variable region (V_(H)) comprises the sequence shownin SEQ ID NO:23, and the 4-1BB agonist light chain variable region(V_(L)) comprises the sequence shown in SEQ ID NO:24, and conservativeamino acid substitutions thereof. In an embodiment, a 4-1BB agonistcomprises V_(H) and V_(L) regions that are each at least 99% identicalto the sequences shown in SEQ ID NO:23 and SEQ ID NO:24, respectively.In an embodiment, a 4-1BB agonist comprises V_(H) and V_(L) regions thatare each at least 98% identical to the sequences shown in SEQ ID NO:23and SEQ ID NO:24, respectively. In an embodiment, a 4-1BB agonistcomprises V_(H) and V_(L) regions that are each at least 97% identicalto the sequences shown in SEQ ID NO:23 and SEQ ID NO:24, respectively.In an embodiment, a 4-1BB agonist comprises V_(H) and V_(L) regions thatare each at least 96% identical to the sequences shown in SEQ ID NO:23and SEQ ID NO:24, respectively. In an embodiment, a 4-1BB agonistcomprises V_(H) and V_(L) regions that are each at least 95% identicalto the sequences shown in SEQ ID NO:23 and SEQ ID NO:24, respectively.In an embodiment, a 4-1BB agonist comprises an scFv antibody comprisingV_(H) and V_(L) regions that are each at least 99% identical to thesequences shown in SEQ ID NO:23 and SEQ ID NO:24.

In an embodiment, a 4-1BB agonist comprises heavy chain CDR1, CDR2 andCDR3 domains having the sequences set forth in SEQ ID NO:25, SEQ IDNO:26, and SEQ ID NO:27, respectively, and conservative amino acidsubstitutions thereof, and light chain CDR1, CDR2 and CDR3 domainshaving the sequences set forth in SEQ ID NO:28, SEQ ID NO:29, and SEQ IDNO:30, respectively, and conservative amino acid substitutions thereof.

In an embodiment, the 4-1BB agonist is a 4-1BB agonist biosimilarmonoclonal antibody approved by drug regulatory authorities withreference to urelumab. In an embodiment, the biosimilar monoclonalantibody comprises an 4-1BB antibody comprising an amino acid sequencewhich has at least 97% sequence identity, e.g., 97%, 98%, 99% or 100%sequence identity, to the amino acid sequence of a reference medicinalproduct or reference biological product and which comprises one or morepost-translational modifications as compared to the reference medicinalproduct or reference biological product, wherein the reference medicinalproduct or reference biological product is urelumab. In someembodiments, the one or more post-translational modifications areselected from one or more of: glycosylation, oxidation, deamidation, andtruncation. In some embodiments, the biosimilar is a 4-1BB agonistantibody authorized or submitted for authorization, wherein the 4-1BBagonist antibody is provided in a formulation which differs from theformulations of a reference medicinal product or reference biologicalproduct, wherein the reference medicinal product or reference biologicalproduct is urelumab. The 4-1BB agonist antibody may be authorized by adrug regulatory authority such as the U.S. FDA and/or the EuropeanUnion's EMA. In some embodiments, the biosimilar is provided as acomposition which further comprises one or more excipients, wherein theone or more excipients are the same or different to the excipientscomprised in a reference medicinal product or reference biologicalproduct, wherein the reference medicinal product or reference biologicalproduct is urelumab. In some embodiments, the biosimilar is provided asa composition which further comprises one or more excipients, whereinthe one or more excipients are the same or different to the excipientscomprised in a reference medicinal product or reference biologicalproduct, wherein the reference medicinal product or reference biologicalproduct is urelumab.

TABLE 8 Amino acid sequences for 4-1BB agonist antibodies related tourelumab. Identifier Sequence (One-Letter Amino Acid Symbols) SEQ ID NO:21 QVQLQQWGAG LLKPSETLSL TCAVYGGSFS GYYWSWIRQS PEKGLEWIGE INHGGYVTYN 60heavy chain for PSLESRVTIS VDTSKNQFSL KLSSVTAADT AVYYCARDYG PGNYDWYFDLWGRGTLVTVS 120 urelumab SASTKGPSVF PLAPCSRSTS ESTAALGCLV KDYFPEPVTVSWNSGALTSG VHTFPAVLQS 180 SGLYSLSSVV TVPSSSLGTK TYTCNVDHKP SNTKVDKRVESKYGPPCPPC PAPEFLGGPS 240 VFLFPPKPKD TLMISRTPEV TCVVVDVSQE DPEVQFNWYVDGVEVHNAKT KPREEQFNST 300 YRVVSVLTVL HQDWLNGKEY KCKVSNKGLP SSIEKTISKAKGQPREPQVY TLPPSQEEMT 360 KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLDSDGSFFLYSR LTVDKSRWQE 420 GNVFSCSVMH EALHNHYTQK SLSLSLGK 448 SEQ ID NO:22 EIVLTQSPAT LSLSPGERAT LSCRASQSVS SYLAWYQQKP GQAPRLLIYD ASNRATGIPA 60light chain for RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ RSNWPPALTF CGGTKVEIKRTVAAPSVFIF 120 urelumab PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGNSQESVTEQDS KDSTYSLSST 180 LTLSKADYEK HKVYACEVTH QGLSSPVTKS FNRGEC 216SEQ ID NO: 23 MKHLWFFLLL VAAPRWVLSQ VQLQQWGAGL LKPSETLSLT CAVYGGSFSGYYWSWIRQSP 60 variable heavy EKGLEWIGEI NHGGYVTYNP SLESRVTISV DTSKNQFSLKLSSVTAADTA VYYCARDYGP 120 chain for urelumab SEQ ID NO: 24 MEAPAQLLFLLLLWLPDTTG EIVLTQSPAT LSLSPGERAT LSCRASQSVS SYLAWYQQKP 60 variable lightGQAPRLLIYD ASNRATGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ 110 chain forurelumab SEQ ID NO: 25 GYYWS 5 heavy chain CDR1 for urelumab SEQ ID NO:26 EINHGGYVTY NPSLES 16 heavy chain CDR2 for urelumab SEQ ID NO: 27DYGPGNYDWY FDL 13 heavy chain CDR3 for urelumab SEQ ID NO: 28 RASQSVSSYLA 11 light chain CDR1 for urelumab SEQ ID NO: 29 DASNRAT 7 light chainCDR2 for urelumab SEQ ID NO: 30 QQRSDWPPAL T 11 light chain CDR3 forurelumab

In an embodiment, the 4-1BB agonist is selected from the groupconsisting of 1D8, 3Elor, 4B4 (BioLegend 309809), H4-1BB-M127 (BDPharmingen 552532), BBK2 (Thermo Fisher MS621PABX), 145501 (LeincoTechnologies B591), the antibody produced by cell line deposited as ATCCNo. HB-11248 and disclosed in U.S. Pat. No. 6,974,863, 5F4 (BioLegend 311503), C65-485 (BD Pharmingen 559446), antibodies disclosed in U.S.Patent Application Publication No. US 2005/0095244, antibodies disclosedin U.S. Pat. No. 7,288,638 (such as 20H4.9-IgG1 (BMS-663031)),antibodies disclosed in U.S. Pat. No. 6,887,673 (such as 4E9 orBMS-554271), antibodies disclosed in U.S. Pat. No. 7,214,493, antibodiesdisclosed in U.S. Pat. No. 6,303,121, antibodies disclosed in U.S. Pat.No. 6,569,997, antibodies disclosed in U.S. Pat. No. 6,905,685 (such as4E9 or BMS-554271), antibodies disclosed in U.S. Pat. No. 6,362,325(such as 1D8 or BMS-469492; 3H3 or BMS-469497; or 3E1), antibodiesdisclosed in U.S. Pat. No. 6,974,863 (such as 53A2); antibodiesdisclosed in U.S. Pat. No. 6,210,669 (such as 1D8, 3B8, or 3E1),antibodies described in U.S. Pat. No. 5,928,893, antibodies disclosed inU.S. Pat. No. 6,303,121, antibodies disclosed in U.S. Pat. No.6,569,997, antibodies disclosed in International Patent ApplicationPublication Nos. WO 2012/177788, WO 2015/119923, and WO 2010/042433, andfragments, derivatives, conjugates, variants, or biosimilars thereof,wherein the disclosure of each of the foregoing patents or patentapplication publications is incorporated by reference here.

In an embodiment, the 4-1BB agonist is a 4-1BB agonistic fusion proteindescribed in International Patent Application Publication Nos. WO2008/025516 A1, WO 2009/007120 A1, WO 2010/003766 A1, WO 2010/010051 A1,and WO 2010/078966 A1; U.S. Patent Application Publication Nos. US2011/0027218 A1, US 2015/0126709 A1, US 2011/0111494 A1, US 2015/0110734A1, and US 2015/0126710 A1; and U.S. Pat. Nos. 9,359,420, 9,340,599,8,921,519, and 8,450,460, the disclosures of which are incorporated byreference herein.

In an embodiment, the 4-1BB agonist is a 4-1BB agonistic fusion proteinas depicted in Structure I-A (C-terminal Fc-antibody fragment fusionprotein) or Structure I-B (N-terminal Fc-antibody fragment fusionprotein), or a fragment, derivative, conjugate, variant, or biosimilarthereof:

In structures I-A and I-B, the cylinders refer to individual polypeptidebinding domains. Structures I-A and I-B comprise three linearly-linkedTNFRSF binding domains derived from e.g., 4-1BBL or an antibody thatbinds 4-1BB, which fold to form a trivalent protein, which is thenlinked to a second triavelent protein through IgG1-Fc (including C_(H)3and C_(H)2 domains) is then used to link two of the trivalent proteinstogether through disulfide bonds (small elongated ovals), stabilizingthe structure and providing an agonists capable of bringing together theintracellular signaling domains of the six receptors and signalingproteins to form a signaling complex. The TNFRSF binding domains denotedas cylinders may be scFv domains comprising, e.g., a V_(H) and a V_(L)chain connected by a linker that may comprise hydrophilic residues andGly and Ser sequences for flexibility, as well as Glu and Lys forsolubility. Any scFv domain design may be used, such as those describedin de Marco, Microbial Cell Factories, 2011, 10, 44; Ahmad, et al.,Clin. & Dev. Immunol. 2012, 980250; Monnier, et al., Antibodies, 2013,2, 193-208; or in references incorporated elsewhere herein. Fusionprotein structures of this form are described in U.S. Pat. Nos.9,359,420, 9,340,599, 8,921,519, and 8,450,460, the disclosures of whichare incorporated by reference herein.

Amino acid sequences for the other polypeptide domains of structure I-Aare given in Table 9. The Fc domain preferably comprises a completeconstant domain (amino acids 17-230 of SEQ ID NO:31) the complete hingedomain (amino acids 1-16 of SEQ ID NO:31) or a portion of the hingedomain (e.g., amino acids 4-16 of SEQ ID NO:31). Preferred linkers forconnecting a C-terminal Fc-antibody may be selected from the embodimentsgiven in SEQ ID NO:32 to SEQ ID NO:41, including linkers suitable forfusion of additional polypeptides.

TABLE 9 Amino acid sequences for TNFRSF fusion proteins, including 4-1BBfusion proteins, with C-terminal Fc-antibody fragment fusion proteindesign (structure I-A). Identifier Sequence (One-Letter Amino AcidSymbols) SEQ ID NO: 31 KSCDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTPEVTCVVVDVS HEDPEVKFNW 60 Fc domain YVDGVEVHNA KTKPREEQYN STYRVVSVLTVLHQDWLNGK EYKCKVSNKA LPAPIEKTIS 120 KAKGQPREPQ VYTLPPSREE MTKNQVSLTCLVKGFYPSDI AVEWESNGQP ENNYKTTPPV 180 LDSDGSFFLY SKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPGK 230 SEQ ID NO: 32 GGPGSSKSCD KTHTCPPCPA PE 22linker SEQ ID NO: 33 GGSGSSKSCD KTHTCPPCPA PE 22 linker SEQ ID NO: 34GGPGSSSSSS SKSCDKTHTC PPCPAPE 27 linker SEQ ID NO: 35 GGSGSSSSSSSKSCDKTHTC PPCPAPE 27 linker SEQ ID NO: 36 GGPGSSSSSS SSSKSCDKTHTCPPCPAPE 29 linker SEQ ID NO: 37 GGSGSSSSSS SSSKSCDKTH TCPPCPAPE 29linker SEQ ID NO: 38 GGPGSSGSGS SDKTHTCPPC PAPE 24 linker SEQ ID NO: 39GGPGSSGSGS DKTHTCPPCP APE 23 linker SEQ ID NO: 40 GGPSSSGSDK THTCPPCPAPE 21 linker SEQ ID NO: 41 GGSSSSSSSS GSDKTHTCPP CPAPE 25 linker

Amino acid sequences for the other polypeptide domains of structure I-Bare given in Table 10. If an Fc antibody fragment is fused to theN-terminus of an TNRFSF fusion protein as in structure I-B, the sequenceof the Fc module is preferably that shown in SEQ ID NO:42, and thelinker sequences are preferably selected from those embodiments setforth in SEQ ID NO:43 to SEQ ID NO:45.

TABLE 10 Amino acid sequences for TNFRSF fusion proteins, including4-1BB fusion proteins, with N-terminal Fc-antibody fragment fusionprotein design (structure I-B). Identifier Sequence (One-Letter AminoAcid Symbols) SEQ ID NO: 42 METDTLLLWV LLLWVPAGNG DKTHTCPPCP APELLGGPSVFLFPPKPKDT LMISRTPEVT 60 Fc domain CVVVDVSHED PEVKFNWYVD GVEVHNAKTKPREEQYNSTY RVVSVLTVLH QDWLNGKEYK 120 CKVSNKALPA PIEKTISKAK GQPREPQVYTLPPSREEMTK NQVSLTCLVK GFYPSDIAVE 180 WESNGQPENN YKTTPPVLDS DGSFFLYSKLTVDKSRWQQG NVFSCSVMHE ALHNHYTQKS 240 LSLSPG 246 SEQ ID NO: 43 SGSGSGSGSGS 11 linker SEQ ID NO: 44 SSSSSSGSGS GS 12 linker SEQ ID NO: 45SSSSSSGSGS GSGSGS 16 linker

In an embodiment, a 4-1BB agonist fusion protein according to structuresI-A or I-B comprises one or more 4-1BB binding domains selected from thegroup consisting of a variable heavy chain and variable light chain ofutomilumab, a variable heavy chain and variable light chain of urelumab,a variable heavy chain and variable light chain of utomilumab, avariable heavy chain and variable light chain selected from the variableheavy chains and variable light chains described in Table 9, anycombination of a variable heavy chain and variable light chain of theforegoing, and fragments, derivatives, conjugates, variants, andbiosimilars thereof.

In an embodiment, a 4-1BB agonist fusion protein according to structuresI-A or I-B comprises one or more 4-1BB binding domains comprising a4-1BBL sequence. In an embodiment, a 4-1BB agonist fusion proteinaccording to structures I-A or I-B comprises one or more 4-1BB bindingdomains comprising a sequence according to SEQ ID NO:46. In anembodiment, a 4-1BB agonist fusion protein according to structures I-Aor I-B comprises one or more 4-1BB binding domains comprising a soluble4-1BBL sequence. In an embodiment, a 4-1BB agonist fusion proteinaccording to structures I-A or I-B comprises one or more 4-1BB bindingdomains comprising a sequence according to SEQ ID NO:47.

In an embodiment, a 4-1BB agonist fusion protein according to structuresI-A or I-B comprises one or more 4-1BB binding domains that is a scFvdomain comprising V_(H) and V_(L) regions that are each at least 95%identical to the sequences shown in SEQ ID NO:13 and SEQ ID NO:14,respectively, wherein the V_(H) and V_(L) domains are connected by alinker. In an embodiment, a 4-1BB agonist fusion protein according tostructures I-A or I-B comprises one or more 4-1BB binding domains thatis a scFv domain comprising V_(H) and V_(L) regions that are each atleast 95% identical to the sequences shown in SEQ ID NO:23 and SEQ IDNO:24, respectively, wherein the V_(H) and V_(L) domains are connectedby a linker. In an embodiment, a 4-1BB agonist fusion protein accordingto structures I-A or I-B comprises one or more 4-1BB binding domainsthat is a scFv domain comprising V_(H) and V_(L) regions that are eachat least 95% identical to the V_(H) and V_(L) sequences given in Table11, wherein the V_(H) and V_(L) domains are connected by a linker.

TABLE 11 Additional polypeptide domains useful as 4-1BB binding domainsin fusion proteins or as scFv 4-1BB agonist antibodies. IdentifierSequence (One-Letter Amino Acid Symbols) SEQ ID NO: 46 MEYASDASLDPEAPWPPAPR ARACRVLPWA LVAGLLLLLL LAAACAVFLA CPWAVSGARA 60 4-1BBLSPGSAASPRL REGPELSPDD PAGLLDLRQG MFAQLVAQNV LLIDGPLSWY SDPGLAGVSL 120TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA 180LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV 240TPEIPAGLPS PRSE 254 SEQ ID NO: 47 LRQGMFAQLV AQNVLLIDGP LSWYSDPGLAGVSLTGGLSY KEDTKELVVA KAGVYYVFFQ 60 4-1BBL soluble LELRRVVAGE GSGSVSLALHLQPLRSAAGA AALALTVDLP PASSEARNSA FGFQGRLLHL 120 domain SAGQRLGVHLHTEARARHAW QLTQGATVLG LFRVTPEIPA GLPSPRSE 168 SEQ ID NO: 48 QVQLQQPGAELVKPGASVKL SCKASGYTFS SYWMHWVKQR PGQVLEWIGE INPGNGHTNY 60 variable heavyNEKFKSKATL TVDKSSSTAY MQLSSLTSED SAVYYCARSF TTARGFAYWG QGTLVTVS 118chain for 4B4-1- 1 version 1 SEQ ID NO: 49 DIVMTQSPAT QSVTPGDRVSLSCRASQTIS DYLHWYQQKS HESPRLLIKY ASQSISGIPS 60 variable light RFSGSGSGSDFTLSINSVEP EDVGVYYCQD GHSFPPTFGG GTKLEIK 107 chain for 4B4-1- 1 version1 SEQ ID NO: 50 QVQLQQPGAE LVKPGASVKL SCKASGYTFS SYWMHWVKQR PGQVLEWIGEINPGNGHTNY 60 variable heavy NEKFKSKATL TVDKSSSTAY MQLSSLTSED SAVYYCARSFTTARGFAYWG QGTLVTVSA 119 chain for 4B4-1- 1 version 2 SEQ ID NO: 51DIVMTQSPAT QSVTPGDRVS LSCRASQTIS DYLHWYQQKS HESPRLLIKY ASQSISGIPS 60variable light RFSGSGSGSD FTLSINSVEP EDVGVYYCQD GHSFPPTFGG GTKLEIKR 108chain for 4B4-1- 1 version 2 SEQ ID NO: 52 MDWTWRILFL VAAATGAHSEVQLVESGGGL VQPGGSLRLS CAASGFTFSD YWMSWVRQAP 60 variable heavy GKGLEWVADIKNDGSYTNYA PSLTNRFTIS RDNAKNSLYL QMNSLRAEDT AVYYCARELT 120 chain forH39E3-2 SEQ ID NO: 53 MEAPAQLLFL LLLWLPDTTG DIVMTQSPDS LAVSLGERATINCKSSQSLL SSGNQKNYL 60 variable light WYQQKPGQPP KLLITYASTR QSGVPDRFSGSGSGTDFTLT ISSLQAEDVA 110 chain for H39E3-2

In an embodiment, the 4-1BB agonist is a 4-1BB agonistic single-chainfusion polypeptide comprising (i) a first soluble 4-1BB binding domain,(ii) a first peptide linker, (iii) a second soluble 4-1BB bindingdomain, (iv) a second peptide linker, and (v) a third soluble 4-1BBbinding domain, further comprising an additional domain at theN-terminal and/or C-terminal end, and wherein the additional domain is aFab or Fc fragment domain. In an embodiment, the 4-1BB agonist is a4-1BB agonistic single-chain fusion polypeptide comprising (i) a firstsoluble 4-1BB binding domain, (ii) a first peptide linker, (iii) asecond soluble 4-1BB binding domain, (iv) a second peptide linker, and(v) a third soluble 4-1BB binding domain, further comprising anadditional domain at the N-terminal and/or C-terminal end, wherein theadditional domain is a Fab or Fc fragment domain, wherein each of thesoluble 4-1BB domains lacks a stalk region (which contributes totrimerisation and provides a certain distance to the cell membrane, butis not part of the 4-1BB binding domain) and the first and the secondpeptide linkers independently have a length of 3-8 amino acids.

In an embodiment, the 4-1BB agonist is a 4-1BB agonistic single-chainfusion polypeptide comprising (i) a first soluble tumor necrosis factor(TNF) superfamily cytokine domain, (ii) a first peptide linker, (iii) asecond soluble TNF superfamily cytokine domain, (iv) a second peptidelinker, and (v) a third soluble TNF superfamily cytokine domain, whereineach of the soluble TNF superfamily cytokine domains lacks a stalkregion and the first and the second peptide linkers independently have alength of 3-8 amino acids, and wherein each TNF superfamily cytokinedomain is a 4-1BB binding domain.

In an embodiment, the 4-1BB agonist is a 4-1BB agonistic scFv antibodycomprising any of the foregoing V_(H) domains linked to any of theforegoing V_(L) domains.

In an embodiment, the 4-1BB agonist is BPS Bioscience 4-1BB agonistantibody catalog no. 79097-2, commercially available from BPSBioscience, San Diego, Calif., USA. In an embodiment, the 4-1BB agonistis Creative Biolabs 4-1BB agonist antibody catalog no. MOM-18179,commercially available from Creative Biolabs, Shirley, N.Y., USA.

3. OX40 (CD134) AGONISTS

In an embodiment, the TNFRSF agonist is an OX40 (CD134) agonist. TheOX40 agonist may be any OX40 binding molecule known in the art. The OX40binding molecule may be a monoclonal antibody or fusion protein capableof binding to human or mammalian OX40. The OX40 agonists or OX40 bindingmolecules may comprise an immunoglobulin heavy chain of any isotype(e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3,IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule. The OX40agonist or OX40 binding molecule may have both a heavy and a lightchain. As used herein, the term binding molecule also includesantibodies (including full length antibodies), monoclonal antibodies(including full length monoclonal antibodies), polyclonal antibodies,multispecific antibodies (e.g., bispecific antibodies), human, humanizedor chimeric antibodies, and antibody fragments, e.g., Fab fragments,F(ab′) fragments, fragments produced by a Fab expression library,epitope-binding fragments of any of the above, and engineered forms ofantibodies, e.g., scFv molecules, that bind to OX40. In an embodiment,the OX40 agonist is an antigen binding protein that is a fully humanantibody. In an embodiment, the OX40 agonist is an antigen bindingprotein that is a humanized antibody. In some embodiments, OX40 agonistsfor use in the presently disclosed methods and compositions includeanti-OX40 antibodies, human anti-OX40 antibodies, mouse anti-OX40antibodies, mammalian anti-OX40 antibodies, monoclonal anti-OX40antibodies, polyclonal anti-OX40 antibodies, chimeric anti-OX40antibodies, anti-OX40 adnectins, anti-OX40 domain antibodies, singlechain anti-OX40 fragments, heavy chain anti-OX40 fragments, light chainanti-OX40 fragments, anti-OX40 fusion proteins, and fragments,derivatives, conjugates, variants, or biosimilars thereof. In apreferred embodiment, the OX40 agonist is an agonistic, anti-OX40humanized or fully human monoclonal antibody (i.e., an antibody derivedfrom a single cell line).

In a preferred embodiment, the OX40 agonist or OX40 binding molecule mayalso be a fusion protein. OX40 fusion proteins comprising an Fc domainfused to OX40L are described, for example, in Sadun, et al., J.Immunother. 2009, 182, 1481-89. In a preferred embodiment, a multimericOX40 agonist, such as a trimeric or hexameric OX40 agonist (with threeor six ligand binding domains), may induce superior receptor (OX40L)clustering and internal cellular signaling complex formation compared toan agonistic monoclonal antibody, which typically possesses two ligandbinding domains. Trimeric (trivalent) or hexameric (or hexavalent) orgreater fusion proteins comprising three TNFRSF binding domains andIgG1-Fc and optionally further linking two or more of these fusionproteins are described, e.g., in Gieffers, et al., Mol. CancerTherapeutics 2013, 12, 2735-47.

Agonistic OX40 antibodies and fusion proteins are known to induce strongimmune responses. Curti, et al., Cancer Res. 2013, 73, 7189-98. In apreferred embodiment, the OX40 agonist is a monoclonal antibody orfusion protein that binds specifically to OX40 antigen in a mannersufficient to reduce toxicity. In some embodiments, the OX40 agonist isan agonistic OX40 monoclonal antibody or fusion protein that abrogatesantibody-dependent cellular toxicity (ADCC), for example NK cellcytotoxicity. In some embodiments, the OX40 agonist is an agonistic OX40monoclonal antibody or fusion protein that abrogates antibody-dependentcell phagocytosis (ADCP). In some embodiments, the OX40 agonist is anagonistic OX40 monoclonal antibody or fusion protein that abrogatescomplement-dependent cytotoxicity (CDC). In some embodiments, the OX40agonist is an agonistic OX40 monoclonal antibody or fusion protein whichabrogates Fc region functionality.

In some embodiments, the OX40 agonists are characterized by binding tohuman OX40 (SEQ ID NO:54) with high affinity and agonistic activity. Inan embodiment, the OX40 agonist is a binding molecule that binds tohuman OX40 (SEQ ID NO:54). In an embodiment, the OX40 agonist is abinding molecule that binds to murine OX40 (SEQ ID NO:55). The aminoacid sequences of OX40 antigen to which an OX40 agonist or bindingmolecule binds are summarized in Table 12.

TABLE 12 Amino acid sequences of OX40 antigens. Identifier Sequence(One-Letter Amino Acid Symbols) SEQ ID NO: 54 MCVGARRLGR GPCAALLLLGLGLSTVTGLH CVGDTYPSND RCCHECRPGN GMVSRCSRSQ 60 human OX40 NTVCRPCGPGFYNDVVSSKP CKPCTWCNLR SGSERKQLCT ATQDTVCRCR AGTQPLDSYK 120 (Homosapiens) PGVDCAPCPP GHFSPGDNQA CKPWTNCTLA GKHTLQPASN SSDAICEDRDPPATQPQETQ 180 GPPARPITVQ PTEAWPRTSQ GPSTRPVEVP GGRAVAAILG LGLVLGLLGPLAILLALYLL 240 RRDQRLPPDA HKPPGGGSFR TPIQEEQADA HSTLAKI 277 SEQ ID NO:55 MYVWVQQPTA LLLLGLTLGV TARRLNCVKH TYPSGHKCCR ECQPGHGMVS RCDHTRDTLC 60murine OX40 HPCETGFYNE AVNYDTCKQC TQCNHRSGSE LKQNCTPTQD TVCRCRPGTQPRQDSGYKLG 120 (Mus musculus) VDCVPCPPGH FSPGNNQACK PWTNCTLSGKQTRHPASDSL DAVCEDRSLL ATLLWETQRP 180 TFRPTTVQST TVWPRTSELP SPPTLVTPEGPAFAVLLGLG LGLLAPLTVL LALYLLRKAW 240 RLPNTPKPCW GNSFRTPIQE EHTDAHFTLA KI272

In some embodiments, the compositions, processes and methods describedinclude a OX40 agonist that binds human or murine OX40 with a K_(D) ofabout 100 pM or lower, binds human or murine OX40 with a K_(D) of about90 pM or lower, binds human or murine OX40 with a K_(D) of about 80 pMor lower, binds human or murine OX40 with a K_(D) of about 70 pM orlower, binds human or murine OX40 with a K_(D) of about 60 pM or lower,binds human or murine OX40 with a K_(D) of about 50 pM or lower, bindshuman or murine OX40 with a K_(D) of about 40 pM or lower, or bindshuman or murine OX40 with a K_(D) of about 30 pM or lower.

In some embodiments, the compositions, processes and methods describedinclude a OX40 agonist that binds to human or murine OX40 with ak_(assoc) of about 7.5×10⁵ 1/M·s or faster, binds to human or murineOX40 with a k_(assoc) of about 7.5×10⁵ 1/M·s or faster, binds to humanor murine OX40 with a k_(assoc) of about 8×10⁵ 1/M·s or faster, binds tohuman or murine OX40 with a k_(assoc) of about 8.5×10⁵ 1/M·s or faster,binds to human or murine OX40 with a k_(assoc) of about 9×10⁵ 1/M·s orfaster, binds to human or murine OX40 with a k_(assoc) of about 9.5×10⁵1/M·s or faster, or binds to human or murine OX40 with a k_(assoc) ofabout 1×10⁶ 1/M·s or faster.

In some embodiments, the compositions, processes and methods describedinclude a OX40 agonist that binds to human or murine OX40 with ak_(dissoc) of about 2×10⁻⁵ 1/s or slower, binds to human or murine OX40with a k_(dissoc) of about 2.1×10⁻⁵ 1/s or slower, binds to human ormurine OX40 with a k_(dissoc) of about 2.2×10⁻⁵ 1/s or slower, binds tohuman or murine OX40 with a k_(dissoc) of about 2.3×10⁻⁵ 1/s or slower,binds to human or murine OX40 with a k_(dissoc) of about 2.4×10⁻⁵ 1/s orslower, binds to human or murine OX40 with a k_(dissoc) of about2.5×10⁻⁵ 1/s or slower, binds to human or murine OX40 with a k_(dissoc)of about 2.6×10⁻⁵1/s or slower or binds to human or murine OX40 with ak_(dissoc) of about 2.7×10⁻⁵ 1/s or slower, binds to human or murineOX40 with a k_(dissoc) of about 2.8×10⁻⁵ 1/s or slower, binds to humanor murine OX40 with a k_(dissoc) of about 2.9×10⁻⁵ 1/s or slower, orbinds to human or murine OX40 with a k_(dissoc) of about 3×10⁻⁵ 1/s orslower.

In some embodiments, the compositions, processes and methods describedinclude OX40 agonist that binds to human or murine OX40 with an IC₅₀ ofabout 10 nM or lower, binds to human or murine OX40 with an IC₅₀ ofabout 9 nM or lower, binds to human or murine OX40 with an IC₅₀ of about8 nM or lower, binds to human or murine OX40 with an IC₅₀ of about 7 nMor lower, binds to human or murine OX40 with an IC₅₀ of about 6 nM orlower, binds to human or murine OX40 with an IC₅₀ of about 5 nM orlower, binds to human or murine OX40 with an IC₅₀ of about 4 nM orlower, binds to human or murine OX40 with an IC₅₀ of about 3 nM orlower, binds to human or murine OX40 with an IC₅₀ of about 2 nM orlower, or binds to human or murine OX40 with an IC₅₀ of about 1 nM orlower.

In some embodiments, the OX40 agonist is tavolixizumab, also known asMEDI0562 or MEDI-0562. Tavolixizumab is available from the MedImmunesubsidiary of AstraZeneca, Inc. Tavolixizumab is immunoglobulinG1-kappa, anti-[Homo sapiens TNFRSF4 (tumor necrosis factor receptor(TNFR) superfamily member 4, OX40, CD134)], humanized and chimericmonoclonal antibody. The amino acid sequences of tavolixizumab are setforth in Table 13. Tavolixizumab comprises N-glycosylation sites atpositions 301 and 301″, with fucosylated complex bi-antennary CHO-typeglycans; heavy chain intrachain disulfide bridges at positions 22-95(V_(H)-V_(L)), 148-204 (C_(H)1-C_(L)), 265-325 (C_(H)2) and 371-429(C_(H)3) (and at positions 22″-95″, 148″-204″, 265″-325″, and371″-429″); light chain intrachain disulfide bridges at positions23′-88′ (V_(H)-V_(L)) and 134′-194′ (C_(H)1-C_(L)) (and at positions23′″-88′″ and 134′-194′″); interchain heavy chain-heavy chain disulfidebridges at positions 230-230″ and 233-233″; and interchain heavychain-light chain disulfide bridges at 224-214′ and 224″-214′. Currentclinical trials of tavolixizumab in a variety of solid tumor indicationsinclude U.S. National Institutes of Health clinicaltrials.govidentifiers NCT02318394 and NCT02705482.

In an embodiment, a OX40 agonist comprises a heavy chain given by SEQ IDNO:56 and a light chain given by SEQ ID NO:57. In an embodiment, a OX40agonist comprises heavy and light chains having the sequences shown inSEQ ID NO:56 and SEQ ID NO:57, respectively, or antigen bindingfragments, Fab fragments, single-chain variable fragments (scFv),variants, or conjugates thereof. In an embodiment, a OX40 agonistcomprises heavy and light chains that are each at least 99% identical tothe sequences shown in SEQ ID NO:56 and SEQ ID NO:57, respectively. Inan embodiment, a OX40 agonist comprises heavy and light chains that areeach at least 98% identical to the sequences shown in SEQ ID NO:56 andSEQ ID NO:57, respectively. In an embodiment, a OX40 agonist comprisesheavy and light chains that are each at least 97% identical to thesequences shown in SEQ ID NO:56 and SEQ ID NO:57, respectively. In anembodiment, a OX40 agonist comprises heavy and light chains that areeach at least 96% identical to the sequences shown in SEQ ID NO:56 andSEQ ID NO:57, respectively. In an embodiment, a OX40 agonist comprisesheavy and light chains that are each at least 95% identical to thesequences shown in SEQ ID NO:56 and SEQ ID NO:57, respectively.

In an embodiment, the OX40 agonist comprises the heavy and light chainCDRs or variable regions (VRs) of tavolixizumab. In an embodiment, theOX40 agonist heavy chain variable region (V_(H)) comprises the sequenceshown in SEQ ID NO:58, and the OX40 agonist light chain variable region(V_(L)) comprises the sequence shown in SEQ ID NO:59, and conservativeamino acid substitutions thereof. In an embodiment, a OX40 agonistcomprises V_(H) and V_(L) regions that are each at least 99% identicalto the sequences shown in SEQ ID NO:58 and SEQ ID NO:59, respectively.In an embodiment, a OX40 agonist comprises V_(H) and V_(L) regions thatare each at least 98% identical to the sequences shown in SEQ ID NO:58and SEQ ID NO:59, respectively. In an embodiment, a OX40 agonistcomprises V_(H) and V_(L) regions that are each at least 97% identicalto the sequences shown in SEQ ID NO:58 and SEQ ID NO:59, respectively.In an embodiment, a OX40 agonist comprises V_(H) and V_(L) regions thatare each at least 96% identical to the sequences shown in SEQ ID NO:58and SEQ ID NO:59, respectively. In an embodiment, a OX40 agonistcomprises V_(H) and V_(L) regions that are each at least 95% identicalto the sequences shown in SEQ ID NO:58 and SEQ ID NO:59, respectively.In an embodiment, an OX40 agonist comprises an scFv antibody comprisingV_(H) and V_(L) regions that are each at least 99% identical to thesequences shown in SEQ ID NO:58 and SEQ ID NO:59.

In an embodiment, a OX40 agonist comprises heavy chain CDR1, CDR2 andCDR3 domains having the sequences set forth in SEQ ID NO:60, SEQ IDNO:61, and SEQ ID NO:62, respectively, and conservative amino acidsubstitutions thereof, and light chain CDR1, CDR2 and CDR3 domainshaving the sequences set forth in SEQ ID NO:63, SEQ ID NO:64, and SEQ IDNO:65, respectively, and conservative amino acid substitutions thereof.

In an embodiment, the OX40 agonist is a OX40 agonist biosimilarmonoclonal antibody approved by drug regulatory authorities withreference to tavolixizumab. In an embodiment, the biosimilar monoclonalantibody comprises an OX40 antibody comprising an amino acid sequencewhich has at least 97% sequence identity, e.g., 97%, 98%, 99% or 100%sequence identity, to the amino acid sequence of a reference medicinalproduct or reference biological product and which comprises one or morepost-translational modifications as compared to the reference medicinalproduct or reference biological product, wherein the reference medicinalproduct or reference biological product is tavolixizumab. In someembodiments, the one or more post-translational modifications areselected from one or more of: glycosylation, oxidation, deamidation, andtruncation. In some embodiments, the biosimilar is a OX40 agonistantibody authorized or submitted for authorization, wherein the OX40agonist antibody is provided in a formulation which differs from theformulations of a reference medicinal product or reference biologicalproduct, wherein the reference medicinal product or reference biologicalproduct is tavolixizumab. The OX40 agonist antibody may be authorized bya drug regulatory authority such as the U.S. FDA and/or the EuropeanUnion's EMA. In some embodiments, the biosimilar is provided as acomposition which further comprises one or more excipients, wherein theone or more excipients are the same or different to the excipientscomprised in a reference medicinal product or reference biologicalproduct, wherein the reference medicinal product or reference biologicalproduct is tavolixizumab. In some embodiments, the biosimilar isprovided as a composition which further comprises one or moreexcipients, wherein the one or more excipients are the same or differentto the excipients comprised in a reference medicinal product orreference biological product, wherein the reference medicinal product orreference biological product is tavolixizumab.

TABLE 13 Amino acid sequences for OX40 agonist antibodies related totavolixizumab. Identifier Sequence (One-Letter Amino Acid Symbols) SEQID NO: 56 QVQLQESGPG LVKPSQTLSL TCAVYGGSFS SGYWNWIRKH PGEGLEYIGYISYNGITYHN 60 heavy chain for PSLKSRITIN RDTSKNQYSL QLNSVTPEDTAVYYCARYKY DYDGGHAMDY WGQGTLVTVS 120 tavolixizumab SASTKGPSVF PLAPSSKSTSGGTAALGCLV KDYFPEPVTV SWNSGALTSG VHTFPAVLQS 180 SGLYSLSSVV TVPSSSLGTQTYICNVNHKP SNTKVDKRVE PKSCDKTHTC PPCPAPELLG 240 GPSVFLFPPK PKDTLMISRTPEVTCVVVDV SHEDPEVKFN WYVDGVEVHN AKTKPREEQY 300 NSTYRVVSVL TVLHQDWLNGKEYKCKVSNK ALPAPIEKTI SKAKGQPREP QVYTLPPSRE 360 EMTKNQVSLT CLVKGFYPSDIAVEWESNGQ PENNYKTTPP VLDSDGSFFL YSKLTVDKSR 420 WQQGNVFSCS VMHEALHNHYTQKSLSLSPG K 451 SEQ ID NO: 57 DIQMTQSPSS LSASVGDRVT ITCRASQDISNYLNWYQQKP GKAPKLLIYY TSKLHSGVPS 60 light chain for RFSGSGSGTDYTLTISSLQP EDFATYYCQQ GSALPWTFGQ GTKVEIKRTV AAPSVFIFPP 120 tavolixizumabSDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT 180LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC 214 SEQ ID NO: 58 QVQLQESGPGLVKPSQTLSL TCAVYGGSFS SGYWNWIRKH PGKGLEYIGY ISYNGITYHN 60 heavy chainPSLKSRITIN RDTSKNQYSL QLNSVTPEDT AVYYCARYKY DYDGGHAMDY WGQGTLVT 118variable region for tavolixizumab SEQ ID NO: 59 DIQMTQSPSS LSASVGDRVTITCRASQDIS NYLNWYQQFP GKAPKLLIYY TSKLHSGVPS 60 light chain RFSGSGSGTDYTLTISSLQP EDFATYYCQQ GSALPWTFGQ GTKVEIKR 108 variable region fortavolixizumab SEQ ID NO: 60 GSFSSGYWN 9 heavy chain CDR1 fortavolixizumab SEQ ID NO: 61 YIGYISYNGI TYH 13 heavy chain CDR2 fortavolixizumab SEQ ID NO: 62 RYKYDYDGGH AMDY 14 heavy chain CDR3 fortavolixizumab SEQ ID NO: 63 QDISNYLN 8 light chain CDR1 fortavolixizumab SEQ ID NO: 64 LLIYYTSELH S 11 light chain CDR2 fortavolixizumab SEQ ID NO: 65 QQGSALPW 8 light chain CDR3 fortavolixizumab

In some embodiments, the OX40 agonist is 11D4, which is a fully humanantibody available from Pfizer, Inc. The preparation and properties of11D4 are described in U.S. Pat. Nos. 7,960,515; 8,236,930; and9,028,824, the disclosures of which are incorporated by referenceherein. The amino acid sequences of 11D4 are set forth in Table 14.

In an embodiment, a OX40 agonist comprises a heavy chain given by SEQ IDNO:66 and a light chain given by SEQ ID NO:67. In an embodiment, a OX40agonist comprises heavy and light chains having the sequences shown inSEQ ID NO:66 and SEQ ID NO:67, respectively, or antigen bindingfragments, Fab fragments, single-chain variable fragments (scFv),variants, or conjugates thereof. In an embodiment, a OX40 agonistcomprises heavy and light chains that are each at least 99% identical tothe sequences shown in SEQ ID NO:66 and SEQ ID NO:67, respectively. Inan embodiment, a OX40 agonist comprises heavy and light chains that areeach at least 98% identical to the sequences shown in SEQ ID NO:66 andSEQ ID NO:67, respectively. In an embodiment, a OX40 agonist comprisesheavy and light chains that are each at least 97% identical to thesequences shown in SEQ ID NO:66 and SEQ ID NO:67, respectively. In anembodiment, a OX40 agonist comprises heavy and light chains that areeach at least 96% identical to the sequences shown in SEQ ID NO:66 andSEQ ID NO:67, respectively. In an embodiment, a OX40 agonist comprisesheavy and light chains that are each at least 95% identical to thesequences shown in SEQ ID NO:66 and SEQ ID NO:67, respectively.

In an embodiment, the OX40 agonist comprises the heavy and light chainCDRs or variable regions (VRs) of 11D4. In an embodiment, the OX40agonist heavy chain variable region (V_(H)) comprises the sequence shownin SEQ ID NO:68, and the OX40 agonist light chain variable region(V_(L)) comprises the sequence shown in SEQ ID NO:69, and conservativeamino acid substitutions thereof. In an embodiment, a OX40 agonistcomprises V_(H) and V_(L) regions that are each at least 99% identicalto the sequences shown in SEQ ID NO:68 and SEQ ID NO:69, respectively.In an embodiment, a OX40 agonist comprises V_(H) and V_(L) regions thatare each at least 98% identical to the sequences shown in SEQ ID NO:68and SEQ ID NO:69, respectively. In an embodiment, a OX40 agonistcomprises V_(H) and V_(L) regions that are each at least 97% identicalto the sequences shown in SEQ ID NO:68 and SEQ ID NO:69, respectively.In an embodiment, a OX40 agonist comprises V_(H) and V_(L) regions thatare each at least 96% identical to the sequences shown in SEQ ID NO:68and SEQ ID NO:69, respectively. In an embodiment, a OX40 agonistcomprises V_(H) and V_(L) regions that are each at least 95% identicalto the sequences shown in SEQ ID NO:68 and SEQ ID NO:69, respectively.

In an embodiment, a OX40 agonist comprises heavy chain CDR1, CDR2 andCDR3 domains having the sequences set forth in SEQ ID NO:70, SEQ IDNO:71, and SEQ ID NO:72, respectively, and conservative amino acidsubstitutions thereof, and light chain CDR1, CDR2 and CDR3 domainshaving the sequences set forth in SEQ ID NO:73, SEQ ID NO:74, and SEQ IDNO:75, respectively, and conservative amino acid substitutions thereof.

In an embodiment, the OX40 agonist is a OX40 agonist biosimilarmonoclonal antibody approved by drug regulatory authorities withreference to 11D4. In an embodiment, the biosimilar monoclonal antibodycomprises an OX40 antibody comprising an amino acid sequence which hasat least 97% sequence identity, e.g., 97%, 98%, 99% or 100% sequenceidentity, to the amino acid sequence of a reference medicinal product orreference biological product and which comprises one or morepost-translational modifications as compared to the reference medicinalproduct or reference biological product, wherein the reference medicinalproduct or reference biological product is 11D4. In some embodiments,the one or more post-translational modifications are selected from oneor more of: glycosylation, oxidation, deamidation, and truncation. Insome embodiments, the biosimilar is a OX40 agonist antibody authorizedor submitted for authorization, wherein the OX40 agonist antibody isprovided in a formulation which differs from the formulations of areference medicinal product or reference biological product, wherein thereference medicinal product or reference biological product is 11D4. TheOX40 agonist antibody may be authorized by a drug regulatory authoritysuch as the U.S. FDA and/or the European Union's EMA. In someembodiments, the biosimilar is provided as a composition which furthercomprises one or more excipients, wherein the one or more excipients arethe same or different to the excipients comprised in a referencemedicinal product or reference biological product, wherein the referencemedicinal product or reference biological product is 11D4. In someembodiments, the biosimilar is provided as a composition which furthercomprises one or more excipients, wherein the one or more excipients arethe same or different to the excipients comprised in a referencemedicinal product or reference biological product, wherein the referencemedicinal product or reference biological product is 11D4.

TABLE 14 Amino acid sequences for OX40 agonist antibodies related to11D4. Identifier Sequence (One-Letter Amino Acid Symbols) SEQ ID NO: 66EVQLVESGGG LVQPGGSLRL SCAASGFTFS SYSMNWVRQA PGKGLEWVSY ISSSSSTIDY 60heavy chain for ADSVKGRFTI SRDNAKNSLY LQMNSLRDED TAVYYCARES GWYLFDYWGQGTLVTVSSAS 120 11D4 TKGPSVFPLA PCSRSTSEST AALGCLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQSSGL 180 YSLSSVVTVP SSNFGTQTYT CNVDHKPSNT KVDKTVERKCCVECPPCPAP PVAGPSVFLF 240 PPKPKDTLMI SRTPEVTCVV VDVSHEDPEV QFNWYVDGVEVHNAKTKPRE EQFNSTFRVV 300 SVLTVVHQDW LNGKEYKCKV SNKGLPAPIE KTISKTKGQPREPQVYTLPP SREEMTKNQV 360 SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPMLDSDGSFFLYSKLTVD KSRWQQGNVF 420 SCSVMHEALH NHYTQKSLSL SPGK 444 SEQ ID NO: 67DIQMTQSPSS LSASVGDRVT ITCRASQGIS SWLAWYQQKP EKAPKSLIYA ASSLQSGVPS 60light chain for RFSGSGSGTD FTLTISSLQP EDFATYYCQQ YNSYPPTFGG GTKVEIKRTVAAPSVFIFPP 120 11D4 SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQESVTEQDSKD STYSLSSTLT 180 LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC 214 SEQID NO: 68 EVQLVESGGG LVQPGGSLRL SCAASGFTFS SYSMNWVRQA PGKGLEWVSYISSSSSTIDY 60 heavy chain ADSVKGRFTI SRDNAKNSLY LQMNSLRDED TAVYYCARESGWYLFDYWGQ GTLVTVSS 118 variable region for 11D4 SEQ ID NO: 69DIQMTQSPSS LSASVGDRVT ITCRASQGIS SWLAWYQQKP EKAPKSLIYA ASSLQSGVPS 60light chain RFSGSGSGTD FTLTISSLQP EDFATYYCQQ YNSYPPTFGG GTKVEIK 107variable region for 11D4 SEQ ID NO: 70 SYSMN 5 heavy chain CDR1 for 11D4SEQ ID NO: 71 YISSSSSTID YADSVKG 17 heavy chain CDR2 for 11D4 SEQ ID NO:72 ESGWYLFDY 9 heavy chain CDR3 for 11D4 SEQ ID NO: 73 RASQGISSWL A 11light chain CDR1 for 11D4 SEQ ID NO: 74 AASSLQS 7 light chain CDR2 for11D4 SEQ ID NO: 75 QQYNSYPPT 9 light chain CDR3 for 11D4

In some embodiments, the OX40 agonist is 18D8, which is a fully humanantibody available from Pfizer, Inc. The preparation and properties of18D8 are described in U.S. Pat. Nos. 7,960,515; 8,236,930; and9,028,824, the disclosures of which are incorporated by referenceherein. The amino acid sequences of 18D8 are set forth in Table 15.

In an embodiment, a OX40 agonist comprises a heavy chain given by SEQ IDNO:76 and a light chain given by SEQ ID NO:77. In an embodiment, a OX40agonist comprises heavy and light chains having the sequences shown inSEQ ID NO:76 and SEQ ID NO:77, respectively, or antigen bindingfragments, Fab fragments, single-chain variable fragments (scFv),variants, or conjugates thereof. In an embodiment, a OX40 agonistcomprises heavy and light chains that are each at least 99% identical tothe sequences shown in SEQ ID NO:76 and SEQ ID NO:77, respectively. Inan embodiment, a OX40 agonist comprises heavy and light chains that areeach at least 98% identical to the sequences shown in SEQ ID NO:76 andSEQ ID NO:77, respectively. In an embodiment, a OX40 agonist comprisesheavy and light chains that are each at least 97% identical to thesequences shown in SEQ ID NO:76 and SEQ ID NO:77, respectively. In anembodiment, a OX40 agonist comprises heavy and light chains that areeach at least 96% identical to the sequences shown in SEQ ID NO:76 andSEQ ID NO:77, respectively. In an embodiment, a OX40 agonist comprisesheavy and light chains that are each at least 95% identical to thesequences shown in SEQ ID NO:76 and SEQ ID NO:77, respectively.

In an embodiment, the OX40 agonist comprises the heavy and light chainCDRs or variable regions (VRs) of 18D8. In an embodiment, the OX40agonist heavy chain variable region (V_(H)) comprises the sequence shownin SEQ ID NO:78, and the OX40 agonist light chain variable region(V_(L)) comprises the sequence shown in SEQ ID NO:79, and conservativeamino acid substitutions thereof. In an embodiment, a OX40 agonistcomprises V_(H) and V_(L) regions that are each at least 99% identicalto the sequences shown in SEQ ID NO:78 and SEQ ID NO:79, respectively.In an embodiment, a OX40 agonist comprises V_(H) and V_(L) regions thatare each at least 98% identical to the sequences shown in SEQ ID NO:78and SEQ ID NO:79, respectively. In an embodiment, a OX40 agonistcomprises V_(H) and V_(L) regions that are each at least 97% identicalto the sequences shown in SEQ ID NO:78 and SEQ ID NO:79, respectively.In an embodiment, a OX40 agonist comprises V_(H) and V_(L) regions thatare each at least 96% identical to the sequences shown in SEQ ID NO:78and SEQ ID NO:79, respectively. In an embodiment, a OX40 agonistcomprises V_(H) and V_(L) regions that are each at least 95% identicalto the sequences shown in SEQ ID NO:78 and SEQ ID NO:79, respectively.

In an embodiment, a OX40 agonist comprises heavy chain CDR1, CDR2 andCDR3 domains having the sequences set forth in SEQ ID NO:80, SEQ IDNO:81, and SEQ ID NO:82, respectively, and conservative amino acidsubstitutions thereof, and light chain CDR1, CDR2 and CDR3 domainshaving the sequences set forth in SEQ ID NO:83, SEQ ID NO:84, and SEQ IDNO:85, respectively, and conservative amino acid substitutions thereof.

In an embodiment, the OX40 agonist is a OX40 agonist biosimilarmonoclonal antibody approved by drug regulatory authorities withreference to 18D8. In an embodiment, the biosimilar monoclonal antibodycomprises an OX40 antibody comprising an amino acid sequence which hasat least 97% sequence identity, e.g., 97%, 98%, 99% or 100% sequenceidentity, to the amino acid sequence of a reference medicinal product orreference biological product and which comprises one or morepost-translational modifications as compared to the reference medicinalproduct or reference biological product, wherein the reference medicinalproduct or reference biological product is 18D8. In some embodiments,the one or more post-translational modifications are selected from oneor more of: glycosylation, oxidation, deamidation, and truncation. Insome embodiments, the biosimilar is a OX40 agonist antibody authorizedor submitted for authorization, wherein the OX40 agonist antibody isprovided in a formulation which differs from the formulations of areference medicinal product or reference biological product, wherein thereference medicinal product or reference biological product is 18D8. TheOX40 agonist antibody may be authorized by a drug regulatory authoritysuch as the U.S. FDA and/or the European Union's EMA. In someembodiments, the biosimilar is provided as a composition which furthercomprises one or more excipients, wherein the one or more excipients arethe same or different to the excipients comprised in a referencemedicinal product or reference biological product, wherein the referencemedicinal product or reference biological product is 18D8. In someembodiments, the biosimilar is provided as a composition which furthercomprises one or more excipients, wherein the one or more excipients arethe same or different to the excipients comprised in a referencemedicinal product or reference biological product, wherein the referencemedicinal product or reference biological product is 18D8.

TABLE 15 Amino acid sequences for OX40 agonist antibodies related to18D8. Identifier Sequence (One-Letter Amino Acid Symbols) SEQ ID NO: 76EVQLVESGGG LVQPGRSLRL SCAASGFTFD DYAMHWVRQA PGKGLEWVSG ISWNSGSIGY 60heavy chain for ADSVKGRFTI SRDNAKNSLY LQMNSLRAED TALYYCAKDQ STADYYFYYGMDVWGQGTTV 120 18D8 TVSSASTKGP SVFPLAPCSR STSESTAALG CLVKDYFPEPVTVSWNSGAL TSGVHTFPAV 180 LQSSGLYSLS SVVTVPSSNF GTQTYTCNVD HKPSNTKVDKTVERKCCVEC PPCPAPPVAG 240 PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVQFNWYVDGVEVHNA KTKPREEQFN 300 STFRVVSVLT VVHQDWLNGK EYKCKVSNKG LPAPIEKTISKTKGQPREPQ VYTLPPSREE 360 MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPMLDSDGSFFLY SKLTVDKSRW 420 QQGNVFSCSV MHEALHNHYT QKSLSLSPGK 450 SEQ IDNO: 77 EIVVTQSPAT LSLSPGERAT LSCRASQSVS SYLAWYQQKP GQAPRLLIYD ASNRATGIPA60 light chain for RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ RSNWPTFGQGTKVEIKRTVA APSVFIFPPS 120 18D8 DEQLKSGTAS VVCLLNNFYP REAKVQWKVDNALQSGNSQE SVTEQDSKDS TYSLSSTLTL 180 SKADYEKHKV YACEVTHQGL SSPVTKSFNRGEC 213 SEQ ID NO: 78 EVQLVESGGG LVQPGRSLRL SCAASGFTFD DYAMHWVRQAPGKGLEWVSG ISWNSGSIGY 60 heavy chain ADSVKGRFTI SRDNAKNSLY LQMNSLRAEDTALYYCAKDQ STADYYFYYG MDVWGQGTTV 120 variable region TVSS 124 for 18D8SEQ ID NO: 79 EIVVTQSPAT LSLSPGERAT LSCRASQSVS SYLAWYQQKP GQAPRLLIYDASNRATGIPA 60 light chain RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ RSNWPTFGQGTKVEIK 106 variable region for 18D8 SEQ ID NO: 80 DYAMH 5 heavy chainCDR1 for 18D8 SEQ ID NO: 81 GISWNSGSIG YADSVKG 17 heavy chain CDR2 for18D8 SEQ ID NO: 82 DQSTADYYFY YGMDV 15 heavy chain CDR3 for 18D8 SEQ IDNO: 83 RASQSVSSYL A 11 light chain CDR1 for 18D8 SEQ ID NO: 84 DASNRAT 7light chain CDR2 for 18D8 SEQ ID NO: 85 QQRSNWPT 8 light chain CDR3 for18D8

In some embodiments, the OX40 agonist is Hu119-122, which is a humanizedantibody available from GlaxoSmithKline plc. The preparation andproperties of Hu119-122 are described in U.S. Pat. Nos. 9,006,399 and9,163,085, and in International Patent Publication No. WO 2012/027328,the disclosures of which are incorporated by reference herein. The aminoacid sequences of Hu119-122 are set forth in Table 16.

In an embodiment, the OX40 agonist comprises the heavy and light chainCDRs or variable regions (VRs) of Hu119-122. In an embodiment, the OX40agonist heavy chain variable region (V_(H)) comprises the sequence shownin SEQ ID NO:86, and the OX40 agonist light chain variable region(V_(L)) comprises the sequence shown in SEQ ID NO:87, and conservativeamino acid substitutions thereof. In an embodiment, a OX40 agonistcomprises V_(H) and V_(L) regions that are each at least 99% identicalto the sequences shown in SEQ ID NO:86 and SEQ ID NO:87, respectively.In an embodiment, a OX40 agonist comprises V_(H) and V_(L) regions thatare each at least 98% identical to the sequences shown in SEQ ID NO:86and SEQ ID NO:87, respectively. In an embodiment, a OX40 agonistcomprises V_(H) and V_(L) regions that are each at least 97% identicalto the sequences shown in SEQ ID NO:86 and SEQ ID NO:87, respectively.In an embodiment, a OX40 agonist comprises V_(H) and V_(L) regions thatare each at least 96% identical to the sequences shown in SEQ ID NO:86and SEQ ID NO:87, respectively. In an embodiment, a OX40 agonistcomprises V_(H) and V_(L) regions that are each at least 95% identicalto the sequences shown in SEQ ID NO:86 and SEQ ID NO:87, respectively.

In an embodiment, a OX40 agonist comprises heavy chain CDR1, CDR2 andCDR3 domains having the sequences set forth in SEQ ID NO:88, SEQ IDNO:89, and SEQ ID NO:90, respectively, and conservative amino acidsubstitutions thereof, and light chain CDR1, CDR2 and CDR3 domainshaving the sequences set forth in SEQ ID NO:91, SEQ ID NO:92, and SEQ IDNO:93, respectively, and conservative amino acid substitutions thereof.

In an embodiment, the OX40 agonist is a OX40 agonist biosimilarmonoclonal antibody approved by drug regulatory authorities withreference to Hu119-122. In an embodiment, the biosimilar monoclonalantibody comprises an OX40 antibody comprising an amino acid sequencewhich has at least 97% sequence identity, e.g., 97%, 98%, 99% or 100%sequence identity, to the amino acid sequence of a reference medicinalproduct or reference biological product and which comprises one or morepost-translational modifications as compared to the reference medicinalproduct or reference biological product, wherein the reference medicinalproduct or reference biological product is Hu119-122. In someembodiments, the one or more post-translational modifications areselected from one or more of: glycosylation, oxidation, deamidation, andtruncation. In some embodiments, the biosimilar is a OX40 agonistantibody authorized or submitted for authorization, wherein the OX40agonist antibody is provided in a formulation which differs from theformulations of a reference medicinal product or reference biologicalproduct, wherein the reference medicinal product or reference biologicalproduct is Hu119-122. The OX40 agonist antibody may be authorized by adrug regulatory authority such as the U.S. FDA and/or the EuropeanUnion's EMA. In some embodiments, the biosimilar is provided as acomposition which further comprises one or more excipients, wherein theone or more excipients are the same or different to the excipientscomprised in a reference medicinal product or reference biologicalproduct, wherein the reference medicinal product or reference biologicalproduct is Hu119-122. In some embodiments, the biosimilar is provided asa composition which further comprises one or more excipients, whereinthe one or more excipients are the same or different to the excipientscomprised in a reference medicinal product or reference biologicalproduct, wherein the reference medicinal product or reference biologicalproduct is Hu119-122.

TABLE 16 Amino acid sequences for OX40 agonist antibodies related toHu119-122. Identifier Sequence (One-Letter Amino Acid Symbols) SEQ IDNO: 86 EVQLVESGGG LVQPGGSLRL SCAASEYEFP SHDMSWVRQA PGKGLELVAA INSDGGSTYY60 heavy chain PDTMERRFTI SRDNAKNSLY LQMNSLRAED TAVYYCARHY DDYYAWFAYWGQGTMVTVSS 120 variable region for Hu119-122 SEQ ID NO: 87 EIVLTQSPATLSLSPGERAT LSCRASKSVS TSGYSYMHWY QQKPGQAPRL LIYLASNLES 60 light chainGVPARFSGSG SGTDFTLTIS SLEPEDFAVY YCQHSRELPL TFGGGTKVEI K 111 variableregion for Hu119-122 SEQ ID NO: 88 SHDMS 5 heavy chain CDR1 forHu119-122 SEQ ID NO: 89 AINSDGGSTY YPDTMER 17 heavy chain CDR2 forHu119-122 SEQ ID NO: 90 HYDDYYAWFA Y 11 heavy chain CDR3 for Hu119-122SEQ ID NO: 91 RASKSVSTSG YSYMH 15 light chain CDR1 for Hu119-122 SEQ IDNO: 92 LASNLES 7 light chain CDR2 for Hu119-122 SEQ ID NO: 93 QHSRELPLT9 light chain CDR3 for Hu119-122

In some embodiments, the OX40 agonist is Hu106-222, which is a humanizedantibody available from GlaxoSmithKline PLC. The preparation andproperties of Hu106-222 are described in U.S. Pat. Nos. 9,006,399 and9,163,085, and in International Patent Publication No. WO 2012/027328,the disclosures of which are incorporated by reference herein. The aminoacid sequences of Hu106-222 are set forth in Table 17.

In an embodiment, the OX40 agonist comprises the heavy and light chainCDRs or variable regions (VRs) of Hu106-222. In an embodiment, the OX40agonist heavy chain variable region (V_(H)) comprises the sequence shownin SEQ ID NO:94, and the OX40 agonist light chain variable region(V_(L)) comprises the sequence shown in SEQ ID NO:95, and conservativeamino acid substitutions thereof. In an embodiment, a OX40 agonistcomprises V_(H) and V_(L) regions that are each at least 99% identicalto the sequences shown in SEQ ID NO:94 and SEQ ID NO:95, respectively.In an embodiment, a OX40 agonist comprises V_(H) and V_(L) regions thatare each at least 98% identical to the sequences shown in SEQ ID NO:94and SEQ ID NO:95, respectively. In an embodiment, a OX40 agonistcomprises V_(H) and V_(L) regions that are each at least 97% identicalto the sequences shown in SEQ ID NO:94 and SEQ ID NO:95, respectively.In an embodiment, a OX40 agonist comprises V_(H) and V_(L) regions thatare each at least 96% identical to the sequences shown in SEQ ID NO:94and SEQ ID NO:95, respectively. In an embodiment, a OX40 agonistcomprises V_(H) and V_(L) regions that are each at least 95% identicalto the sequences shown in SEQ ID NO:94 and SEQ ID NO:95, respectively.

In an embodiment, a OX40 agonist comprises heavy chain CDR1, CDR2 andCDR3 domains having the sequences set forth in SEQ ID NO:96, SEQ IDNO:97, and SEQ ID NO:98, respectively, and conservative amino acidsubstitutions thereof, and light chain CDR1, CDR2 and CDR3 domainshaving the sequences set forth in SEQ ID NO:99, SEQ ID NO:100, and SEQID NO:101, respectively, and conservative amino acid substitutionsthereof.

In an embodiment, the OX40 agonist is a OX40 agonist biosimilarmonoclonal antibody approved by drug regulatory authorities withreference to Hu106-222. In an embodiment, the biosimilar monoclonalantibody comprises an OX40 antibody comprising an amino acid sequencewhich has at least 97% sequence identity, e.g., 97%, 98%, 99% or 100%sequence identity, to the amino acid sequence of a reference medicinalproduct or reference biological product and which comprises one or morepost-translational modifications as compared to the reference medicinalproduct or reference biological product, wherein the reference medicinalproduct or reference biological product is Hu106-222. In someembodiments, the one or more post-translational modifications areselected from one or more of: glycosylation, oxidation, deamidation, andtruncation. In some embodiments, the biosimilar is a OX40 agonistantibody authorized or submitted for authorization, wherein the OX40agonist antibody is provided in a formulation which differs from theformulations of a reference medicinal product or reference biologicalproduct, wherein the reference medicinal product or reference biologicalproduct is Hu106-222. The OX40 agonist antibody may be authorized by adrug regulatory authority such as the U.S. FDA and/or the EuropeanUnion's EMA. In some embodiments, the biosimilar is provided as acomposition which further comprises one or more excipients, wherein theone or more excipients are the same or different to the excipientscomprised in a reference medicinal product or reference biologicalproduct, wherein the reference medicinal product or reference biologicalproduct is Hu106-222. In some embodiments, the biosimilar is provided asa composition which further comprises one or more excipients, whereinthe one or more excipients are the same or different to the excipientscomprised in a reference medicinal product or reference biologicalproduct, wherein the reference medicinal product or reference biologicalproduct is Hu106-222.

TABLE 17 Amino acid sequences for OX40 agonist antibodies related toHu106-222. Identifier Sequence (One-Letter Amino Acid Symbols) SEQ IDNO: 94 QVQLVQSGSE LKKPGASVKV SCKASGYTFT DYSMHWVRQA PGQGLKWMGW INTETGEPTY60 heavy chain ADDFKGRFVF SLDTSVSTAY LQISSLKAED TAVYYCANPY YDYVSYYAMDYWGQGTTVTV 120 variable region SS 122 for Hu106-222 SEQ ID NO: 95DIQMTQSPSS LSASVGDRVT ITCKASQDVS TAVAWYQQKP GKAPKLLIYS ASYLYTGVPS 60light chain RFSGSGSGTD FTFTISSLQP EDIATYYCQQ HYSTPRTFGQ GTKLEIK 107variable region for Hu106-222 SEQ ID NO: 96 DYSMH 5 heavy chain CDR1 forHu106-222 SEQ ID NO: 97 WINTETGEPT YADDFKG 17 heavy chain CDR2 forHu106-222 SEQ ID NO: 98 PYYDYVSYYA MDY 13 heavy chain CDR3 for Hu106-222SEQ ID NO: 99 KASQDVSTAV A 11 light chain CDR1 for Hu106-222 SEQ ID NO:100 SASYLYT 7 light chain CDR2 for Hu106-222 SEQ ID NO: 101 QQHYSTPRT 9light chain CDR3 for Hu106-222

In some embodiments, the OX40 agonist antibody is MEDI6469 (alsoreferred to as 9B12). MEDI6469 is a murine monoclonal antibody.Weinberg, et al., J. Immunother. 2006, 29, 575-585. In some embodimentsthe OX40 agonist is an antibody produced by the 9B12 hybridoma,deposited with Biovest Inc. (Malvern, Mass., USA), as described inWeinberg, et al., J. Immunother. 2006, 29, 575-585, the disclosure ofwhich is hereby incorporated by reference in its entirety. In someembodiments, the antibody comprises the CDR sequences of MEDI6469. Insome embodiments, the antibody comprises a heavy chain variable regionsequence and/or a light chain variable region sequence of MEDI6469.

In an embodiment, the OX40 agonist is L106 BD (Pharmingen Product#340420). In some embodiments, the OX40 agonist comprises the CDRs ofantibody L106 (BD Pharmingen Product #340420). In some embodiments, theOX40 agonist comprises a heavy chain variable region sequence and/or alight chain variable region sequence of antibody L106 (BD PharmingenProduct #340420). In an embodiment, the OX40 agonist is ACT35 (SantaCruz Biotechnology, Catalog #20073). In some embodiments, the OX40agonist comprises the CDRs of antibody ACT35 (Santa Cruz Biotechnology,Catalog #20073). In some embodiments, the OX40 agonist comprises a heavychain variable region sequence and/or a light chain variable regionsequence of antibody ACT35 (Santa Cruz Biotechnology, Catalog #20073).In an embodiment, the OX40 agonist is the murine monoclonal antibodyanti-mCD134/mOX40 (clone OX86), commercially available from InVivoMAb,BioXcell Inc, West Lebanon, N.H.

In an embodiment, the OX40 agonist is selected from the OX40 agonistsdescribed in International Patent Application Publication Nos. WO95/12673, WO 95/21925, WO 2006/121810, WO 2012/027328, WO 2013/028231,WO 2013/038191, and WO 2014/148895; European Patent Application EP0672141; U.S. Patent Application Publication Nos. US 2010/136030, US2014/377284, US 2015/190506, and US 2015/132288 (including clones 20E5and 12H3); and U.S. Pat. Nos. 7,504,101, 7,550,140, 7,622,444,7,696,175, 7,960,515, 7,961,515, 8,133,983, 9,006,399, and 9,163,085,the disclosure of each of which is incorporated herein by reference inits entirety for all purposes and in particular for all teachingsrelated to OX40 agonists and their use.

In an embodiment, the OX40 agonist is an OX40 agonistic fusion proteinas depicted in Structure I-A (C-terminal Fc-antibody fragment fusionprotein) or Structure I-B (N-terminal Fc-antibody fragment fusionprotein), or a fragment, derivative, conjugate, variant, or biosimilarthereof. The properties of structures I-A and I-B are described aboveand in U.S. Pat. Nos. 9,359,420, 9,340,599, 8,921,519, and 8,450,460,the disclosures of which are incorporated by reference herein. Aminoacid sequences for the polypeptide domains of structure I-A are given inTable 9. The Fc domain preferably comprises a complete constant domain(amino acids 17-230 of SEQ ID NO:31) the complete hinge domain (aminoacids 1-16 of SEQ ID NO:31) or a portion of the hinge domain (e.g.,amino acids 4-16 of SEQ ID NO:31). Preferred linkers for connecting aC-terminal Fc-antibody may be selected from the embodiments given in SEQID NO:32 to SEQ ID NO:41, including linkers suitable for fusion ofadditional polypeptides. Likewise, amino acid sequences for thepolypeptide domains of structure I-B are given in Table 10. If an Fcantibody fragment is fused to the N-terminus of an TNRFSF fusion proteinas in structure I-B, the sequence of the Fc module is preferably thatshown in SEQ ID NO:42, and the linker sequences are preferably selectedfrom those embodiments set forth in SED ID NO:43 to SEQ ID NO:45.

In an embodiment, an OX40 agonist fusion protein according to structuresI-A or I-B comprises one or more OX40 binding domains selected from thegroup consisting of a variable heavy chain and variable light chain oftavolixizumab, a variable heavy chain and variable light chain of 11D4,a variable heavy chain and variable light chain of 18D8, a variableheavy chain and variable light chain of Hu119-122, a variable heavychain and variable light chain of Hu106-222, a variable heavy chain andvariable light chain selected from the variable heavy chains andvariable light chains described in Table 17, any combination of avariable heavy chain and variable light chain of the foregoing, andfragments, derivatives, conjugates, variants, and biosimilars thereof.

In an embodiment, an OX40 agonist fusion protein according to structuresI-A or I-B comprises one or more OX40 binding domains comprising anOX40L sequence. In an embodiment, an OX40 agonist fusion proteinaccording to structures I-A or I-B comprises one or more OX40 bindingdomains comprising a sequence according to SEQ ID NO:102. In anembodiment, an OX40 agonist fusion protein according to structures I-Aor I-B comprises one or more OX40 binding domains comprising a solubleOX40L sequence. In an embodiment, a OX40 agonist fusion proteinaccording to structures I-A or I-B comprises one or more OX40 bindingdomains comprising a sequence according to SEQ ID NO:103. In anembodiment, a OX40 agonist fusion protein according to structures I-A orI-B comprises one or more OX40 binding domains comprising a sequenceaccording to SEQ ID NO:104.

In an embodiment, an OX40 agonist fusion protein according to structuresI-A or I-B comprises one or more OX40 binding domains that is a scFvdomain comprising V_(H) and V_(L) regions that are each at least 95%identical to the sequences shown in SEQ ID NO:58 and SEQ ID NO:59,respectively, wherein the V_(H) and V_(L) domains are connected by alinker. In an embodiment, an OX40 agonist fusion protein according tostructures I-A or I-B comprises one or more OX40 binding domains that isa scFv domain comprising V_(H) and V_(L) regions that are each at least95% identical to the sequences shown in SEQ ID NO:68 and SEQ ID NO:69,respectively, wherein the V_(H) and V_(L) domains are connected by alinker. In an embodiment, an OX40 agonist fusion protein according tostructures I-A or I-B comprises one or more OX40 binding domains that isa scFv domain comprising V_(H) and V_(L) regions that are each at least95% identical to the sequences shown in SEQ ID NO:78 and SEQ ID NO:79,respectively, wherein the V_(H) and V_(L) domains are connected by alinker. In an embodiment, an OX40 agonist fusion protein according tostructures I-A or I-B comprises one or more OX40 binding domains that isa scFv domain comprising V_(H) and V_(L) regions that are each at least95% identical to the sequences shown in SEQ ID NO:86 and SEQ ID NO:87,respectively, wherein the V_(H) and V_(L) domains are connected by alinker. In an embodiment, an OX40 agonist fusion protein according tostructures I-A or I-B comprises one or more OX40 binding domains that isa scFv domain comprising V_(H) and V_(L) regions that are each at least95% identical to the sequences shown in SEQ ID NO:94 and SEQ ID NO:95,respectively, wherein the V_(H) and V_(L) domains are connected by alinker. In an embodiment, an OX40 agonist fusion protein according tostructures I-A or I-B comprises one or more OX40 binding domains that isa scFv domain comprising V_(H) and V_(L) regions that are each at least95% identical to the V_(H) and V_(L) sequences given in Table 18,wherein the V_(H) and V_(L) domains are connected by a linker.

TABLE 18 Additional polypeptide domains useful as OX40 binding domainsin fusion proteins (e.g., structures I-A and I-B) or as scFv OX40agonist antibodies. Identifier Sequence (One-Letter Amino Acid Symbols)SEQ ID NO: 102 MERVQPLEEN VGNAARPRFE RNKLLLVASV IQGLGLLLCF TYICLHFSALQVSHRYPRIQ 60 OX40L SIKVQFTEYK KEKGFILTSQ KEDEIMKVQN NSVIINCDGFYLISLKGYFS QEVNISLHYQ 120 KDEEPLFQLK KVRSVNSLMV ASLTYKDKVY LNVTTDNTSLDDFHVNGGEL ILIHQNPGEF 180 CVL 183 SEQ ID NO: 103 SHRYPRIQSI KVQFTEYKKEKGFILTSQKE DEIMKVQNNS VIINCDGFYL ISLKGYFSQE 60 OX40L soluble VNISLHYQKDEEPLFQLKKV RSVNSLMVAS LTYKDKVYLN VTTDNTSLDD FHVNGGELIL 120 domainIHQNPGEFCV L 131 SEQ ID NO: 104 YPRIQSIKVQ FTEYKKEKGF ILTSQKEDEIMKVQNNSVII NCDGFYLISL KGYFSQEVNI 60 OX40L soluble SLHYQKDEEP LFQLKKVRSVNSLMVASLTY KDKVYLNVTT DNTSLDDFHV NGGELILIHQ 120 domain NPGEFCVL 128(alternative) SEQ ID NO: 105 EVQLVESGGG LVQPGGSLRL SCAASGFTFS NYTMNWVRQAPGKGLEWVSA ISGSGGSTYY 60 variable heavy ADSVKGRFTI SRDNSKNTLY LQMNSLRAEDTAVYYCAKDR YSQVHYALDY WGQGTLVTVS 120 chain for 008 SEQ ID NO: 106DIVMTQSPDS LPVTPGEPAS ISCRSSQSLL HSNGYNYLDW YLQKAGQSPQ LLIYLGSNRA 60variable light SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCQQYYNHP TTFGQGTK 108chain for 008 SEQ ID NO: 107 EVQLVESGGG VVQPGRSLRL SCAASGFTFS DYTMNWVRQAPGKGLEWVSS ISGGSTYYAD 60 variable heavy SRKGRFTISR DNSKNTLYLQ MNNLRAEDTAVYYCARDRYF RQQNAFDYWG QGTLVTVSSA 120 chain for 011 SEQ ID NO: 108DIVMTQSPDS LPVTPGEPAS ISCRSSQSLL HSNGYNYLDW YLQKAGQSPQ LLIYLGSNRA 60variable light SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCQQYYNHP TTFGQGTK 108chain for 011 SEQ ID NO: 109 EVQLVESGGG LVQPRGSLRL SCAASGFTFS SYAMNWVRQAPGKGLEWVAV ISYDGSNKYY 60 variable heavy ADSVKGRFTI SRDNSKNTLY LQMNSLRAEDTAVYYCAKDR YITLPNALDY WGQGTLVTVS 120 chain for 021 SEQ ID NO: 110DIQMTQSPVS LPVTPGEPAS ISCRSSQSLL HSNGYNYLDW YLQKPGQSPQ LLIYLGSNRA 60variable light SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCQQYKSNP PTFGQGTK 108chain for 021 SEQ ID NO: 111 EVQLVESGGG LVHPGGSLRL SCAGSGFTFS SYAMHWVRQAPGKGLEWVSA IGTGGGTYYA 60 variable heavy DSVMGRFTIS RDNSKNTLYL QMNSLRAEDTAVYYCARYDN VMGLYWFDYW GQGTLVTVSS 120 chain for 023 SEQ ID NO: 112EIVLTQSPAT LSLSPGERAT LSCRASQSVS SYLAWYQQKP GQAPRLLIYD ASNRATGIPA 60variable light RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ RSNWPPAFGG GTKVEIKR 108chain for 023 SEQ ID NO: 113 EVQLQQSGPE LVKPGASVKM SCKASGYTFT SYVMHWVKQKPGQGLEWIGY INPYNDGTKY 60 heavy chain NEKFKGKATL TSDKSSSTAY MELSSLTSEDSAVYYCANYY GSSLSMDYWG QGTSVTVSS 119 variable region SEQ ID NO: 114DIQMTQTTSS LSASLGDRVT ISCRASQDIS NYLNWYQQKP DGTVKLLIYY TSRLHSGVPS 60light chain RFSGSGSGTD YSLTISNLEQ EDIATYFCQQ GNTLPWTFGG GTKLEIKR 108variable region SEQ ID NO: 115 EVQLQQSGPE LVKPGASVKI SCKTSGYTFKDYTMHWVKQS HGKSLEWIGG IYPNNGGSTY 60 heavy chain NQNFKDKATL TVDKSSSTAYMEFRSLTSED SAVYYCARMG YHGPHLDFDV WGAGTTVTVS 120 variable region P 121SEQ ID NO: 116 DIVMTQSHKF MSTSLGDRVS ITCKASQDVG AAVAWYQQKP GQSPKLLIYWASTRHTGVPD 60 light chain RFTGGGSGTD FTLTISNVQS EDLTDYFCQQ YINYPLTFGGGTKLEIKR 108 variable region SEQ ID NO: 117 QIQLVQSGPE LKKPGETVKISCKASGYTFT DYSMHWVKQA PGKGLKWMGW INTETGEPTY 60 heavy chain ADDFKGRFAFSLETSASTAY LQINNLKNED TATYFCANPY YDYVSYYAMD YWGHGTSVTV 120 variableregion SS 122 of humanized antibody SEQ ID NO: 118 QVQLVQSGSE LKKPGASVKVSCKASGYTFT DYSMHWVRQA PGQGLKWMGW INTETGEPTY 60 heavy chain ADDFKGRFVFSLDTSVSTAY LQISSLKAED TAVYYCANPY YDYVSYYAMD YWGQGTTVTV 120 variableregion SS 122 of humanized antibody SEQ ID NO: 119 DIVMTQSHKF MSTSVRDRVSITCKASQDVS TAVAWYQQKP GQSPKLLIYS ASYLYTGVPD 60 light chain RFTGSGSGTDFTFTISSVQA EDLAVYYCQQ HYSTPRTFGG GTKLEIK 107 variable region ofhumanized antibody SEQ ID NO: 120 DIVMTQSHKF MSTSVRDRVS ITCKASQDVSTAVAWYQQKP GQSPKLLIYS ASYLYTGVPD 60 light chain RFTGSGSGTD FTFTISSVQAEDLAVYYCQQ HYSTPRTFGG GTKLEIK 107 variable region of humanized antibodySEQ ID NO: 121 EVQLVESGGG LVQPGESLKL SCESNEYEFP SHDMSWVRKT PEKRLELVAAINSDGGSTYY 60 heavy chain PDTMERRFII SRDNTKKTLY LQMSSLRSED TALYYCARHYDDYYAWFAYW GQGTLVTVSA 120 variable region of humanized antibody SEQ IDNO: 122 EVQLVESGGG LVQPGGSLRL SCAASEYEFP SHDMSWVRQA PGKGLELVAAINSDGGSTYY 60 heavy chain PDTMERRFTI SRDNAKNSLY LQMNSLRAED TAVYYCARHYDDYYAWFAYW GQGTMVTVSS 120 variable region of humanized antibody SEQ IDNO: 123 DIVLTQSPAS LAVSLGQRAT ISCRASKSVS TSGYSYMHWY QQKPGQPPKLLIYLASNLES 60 light chain GVPARFSGSG SGTDFTLNIH PVEEEDAATY YCQHSRELPLTFGAGTKLEL K 111 variable region of humanized antibody SEQ ID NO: 124EIVLTQSPAT LSLSPGERAT LSCRASKSVS TSGYSYMHWY QQKPGQAPRL LIYLASNLES 60light chain GVPARFSGSG SGTDFTLTIS SLEPEDEAVY YCQHSRELPL TFGGGTKVEI K 111variable region of humanized antibody SEQ ID NO: 125 MYLGLNYVFIVFLLNGVQSE VKLEESGGGL VQPGGSMKLS CAASGFTFSD AWMDWVRQSP 60 heavy chainEKGLEWVAEI RSKANNHATY YAESVNGRFT ISRDDSKSSV YLQMNSLRAE DTGIYYCTWG 120variable region EVFYFDYWGQ GTTLTVSS 138 SEQ ID NO: 126 MRPSIQFLGLLLFWLHGAQC DIQMTQSPSS LSASLGGKVT ITCKSSQDIN KYIAWYQHKP 60 light chainGKGPRLLIHY TSTLQPGIPS RFSGSGSGRD YSFSISNLEP EDIATYYCLQ YDNLLTFGAG 120variable region TKLELK 126

In an embodiment, the OX40 agonist is a OX40 agonistic single-chainfusion polypeptide comprising (i) a first soluble OX40 binding domain,(ii) a first peptide linker, (iii) a second soluble OX40 binding domain,(iv) a second peptide linker, and (v) a third soluble OX40 bindingdomain, further comprising an additional domain at the N-terminal and/orC-terminal end, and wherein the additional domain is a Fab or Fcfragment domain. In an embodiment, the OX40 agonist is a OX40 agonisticsingle-chain fusion polypeptide comprising (i) a first soluble OX40binding domain, (ii) a first peptide linker, (iii) a second soluble OX40binding domain, (iv) a second peptide linker, and (v) a third solubleOX40 binding domain, further comprising an additional domain at theN-terminal and/or C-terminal end, wherein the additional domain is a Fabor Fc fragment domain wherein each of the soluble OX40 binding domainslacks a stalk region (which contributes to trimerisation and provides acertain distance to the cell membrane, but is not part of the OX40binding domain) and the first and the second peptide linkersindependently have a length of 3-8 amino acids.

In an embodiment, the OX40 agonist is an OX40 agonistic single-chainfusion polypeptide comprising (i) a first soluble tumor necrosis factor(TNF) superfamily cytokine domain, (ii) a first peptide linker, (iii) asecond soluble TNF superfamily cytokine domain, (iv) a second peptidelinker, and (v) a third soluble TNF superfamily cytokine domain, whereineach of the soluble TNF superfamily cytokine domains lacks a stalkregion and the first and the second peptide linkers independently have alength of 3-8 amino acids, and wherein the TNF superfamily cytokinedomain is an OX40 binding domain.

In some embodiments, the OX40 agonist is MEDI6383 MEDI6383 is an OX40agonistic fusion protein and can be prepared as described in U.S. Pat.No. 6,312,700, the disclosure of which is incorporated by referenceherein.

In an embodiment, the OX40 agonist is an OX40 agonistic scFv antibodycomprising any of the foregoing V_(H) domains linked to any of theforegoing V_(L) domains.

In an embodiment, the OX40 agonist is Creative Biolabs OX40 agonistmonoclonal antibody MOM-18455, commercially available from CreativeBiolabs, Inc., Shirley, N.Y., USA.

In an embodiment, the OX40 agonist is OX40 agonistic antibody cloneBer-ACT35 commercially available from BioLegend, Inc., San Diego,Calif., USA.

Optional Cell Viability Analyses

Optionally, a cell viability assay can be performed after the firstexpansion (sometimes referred to as the initial bulk expansion), usingstandard assays known in the art. For example, a trypan blue exclusionassay can be done on a sample of the bulk TILs, which selectively labelsdead cells and allows a viability assessment. Other assays for use intesting viability can include but are not limited to the Alamar blueassay; and the MTT assay.

1. Cell Counts, Viability, Flow Cytometry

In some embodiments, cell counts and/or viability are measured. Theexpression of markers such as but not limited CD3, CD4, CD8, and CD56,as well as any other disclosed or described herein, can be measured byflow cytometry with antibodies, for example but not limited to thosecommercially available from BD Bio-sciences (BD Biosciences, San Jose,Calif.) using a FACSCanto™ flow cytometer (BD Biosciences). The cellscan be counted manually using a disposable c-chip hemocytometer (VWR,Batavia, Ill.) and viability can be assessed using any method known inthe art, including but not limited to trypan blue staining. The cellviability can also be assayed based on U.S. Ser. No. 15/863,634,incorporated by reference herein in its entirety.

In some cases, the bulk TIL population can be cryopreserved immediately,using the protocols discussed below. Alternatively, the bulk TILpopulation can be subjected to REP and then cryopreserved as discussedbelow. Similarly, in the case where genetically modified TILs will beused in therapy, the bulk or REP TIL populations can be subjected togenetic modifications for suitable treatments.

According to the present disclosure, a method for assaying TILs forviability and/or further use in administration to a subject. In someembodiments, the method for assay tumor infiltrating lymphocytes (TILs)comprises:

-   -   (i) obtaining a first population of TILs;    -   (ii) performing a first expansion by culturing the first        population of TILs in a cell culture medium comprising IL-2, and        optionally OKT-3, to produce a second population of TILs; and    -   (iii) performing a second expansion by supplementing the cell        culture medium of the second population of TILs with additional        IL-2, OKT-3, and antigen presenting cells (APCs), to produce a        third population of TILs, wherein the third population of TILs        is at least 50-fold greater in number than the second population        of TILs;    -   (iv) harvesting, washing, and cryopreserving the third        population of TILs;    -   (v) storing the cryopreserved TILs at a cryogenic temperature;    -   (vi) thawing the third population of TILs to provide a thawed        third population of TILs; and    -   (vii) performing an additional second expansion of a portion of        the thawed third population of TILs by supplementing the cell        culture medium of the third population with IL-2, OKT-3, and        APCs for an additional expansion period (sometimes referred to        as a reREP period) of at least 3 days, wherein the third        expansion is performed to obtain a fourth population of TILs,        wherein the number of TILs in the fourth population of TILs is        compared to the number of TILs in the third population of TILs        to obtain a ratio;    -   (viii) determining based on the ratio in step (vii) whether the        thawed population of TILs is suitable for administration to a        patient;    -   (ix) administering a therapeutically effective dosage of the        thawed third population of TILs to the patient when the ratio of        the number of TILs in the fourth population of TILs to the        number of TILs in the third population of TILs is determined to        be greater than 5:1 in step (viii).

In some embodiments, the TILs are assayed for viability after step(vii).

The present disclosure also provides further methods for assaying TILs.In some embodiments, the disclosure provides a method for assaying TILscomprising:

-   -   (i) obtaining a portion of a first population of cryopreserved        TILs;    -   (ii) thawing the portion of the first population of        cryopreserved TILs;    -   (iii) performing a first expansion by culturing the portion of        the first population of TILs in a cell culture medium comprising        IL-2, OKT-3, and antigen presenting cells (APCs) for an        additional expansion period (sometimes referred to as a reREP        period) of at least 3 days, to produce a second population of        TILs, wherein the portion from the first population of TILs is        compared to the second population of TILs to obtain a ratio of        the number of TILs, wherein the ratio of the number of TILs in        the second population of TILs to the number of TILs in the        portion of the first population of TILs is greater than 5:1;    -   (iv) determining based on the ratio in step (iii) whether the        first population of TILs is suitable for use in therapeutic        administration to a patient;    -   (v) determining the first population of TILs is suitable for use        in therapeutic administration when the ratio of the number of        TILs in the second population of TILs to the number of TILs in        the first population of TILs is determined to be greater than        5:1 in step (iv).

In some embodiments, the ratio of the number of TILs in the secondpopulation of TILs to the number of TILs in the portion of the firstpopulation of TILs is greater than 50:1.

In some embodiments, the method further comprises performing expansionof the entire first population of cryopreserved TILs from step (i)according to the methods as described in any of the embodiments providedherein.

In some embodiments, the method further comprises administering theentire first population of cryopreserved TILs from step (i) to thepatient.

2. Cell Cultures

In an embodiment, a method for expanding TILs, including those discussedabove as well as exemplified in FIG. 18, may include using about 5,000mL to about 25,000 mL of cell medium, about 5,000 mL to about 10,000 mLof cell medium, or about 5,800 mL to about 8,700 mL of cell medium. Insome embodiments, the media is a serum free medium. In some embodiments,the media in the first expansion is serum free. In some embodiments, themedia in the second expansion is serum free. In some embodiments, themedia in the first expansion and the second are both serum free. In anembodiment, expanding the number of TILs uses no more than one type ofcell culture medium. Any suitable cell culture medium may be used, e.g.,AIM-V cell medium (L-glutamine, 50 μM streptomycin sulfate, and 10 μMgentamicin sulfate) cell culture medium (Invitrogen, Carlsbad Calif.).In this regard, the inventive methods advantageously reduce the amountof medium and the number of types of medium required to expand thenumber of TIL. In an embodiment, expanding the number of TIL maycomprise feeding the cells no more frequently than every third or fourthday. Expanding the number of cells in a gas permeable containersimplifies the procedures necessary to expand the number of cells byreducing the feeding frequency necessary to expand the cells.

In an embodiment, the cell medium in the first and/or second gaspermeable container is unfiltered. The use of unfiltered cell medium maysimplify the procedures necessary to expand the number of cells. In anembodiment, the cell medium in the first and/or second gas permeablecontainer lacks beta-mercaptoethanol (BME).

In an embodiment, the duration of the method comprising obtaining atumor tissue sample from the mammal; culturing the tumor tissue samplein a first gas permeable container containing cell medium therein;obtaining TILs from the tumor tissue sample; expanding the number ofTILs in a second gas permeable container containing cell medium for aduration of about 7 to 14 days, e.g., about 11 days. In some embodimentspre-REP is about 7 to 14 days, e.g., about 11 days. In some embodiments,REP is about 7 to 14 days, e.g., about 11 days.

In an embodiment, TILs are expanded in gas-permeable containers.Gas-permeable containers have been used to expand TILs using PBMCs usingmethods, compositions, and devices known in the art, including thosedescribed in U.S. Patent Application Publication No. 2005/0106717 A1,the disclosures of which are incorporated herein by reference. In anembodiment, TILs are expanded in gas-permeable bags. In an embodiment,TILs are expanded using a cell expansion system that expands TILs in gaspermeable bags, such as the Xuri Cell Expansion System W25 (GEHealthcare). In an embodiment, TILs are expanded using a cell expansionsystem that expands TILs in gas permeable bags, such as the WAVEBioreactor System, also known as the Xuri Cell Expansion System W5 (GEHealthcare). In an embodiment, the cell expansion system includes a gaspermeable cell bag with a volume selected from the group consisting ofabout 100 mL, about 200 mL, about 300 mL, about 400 mL, about 500 mL,about 600 mL, about 700 mL, about 800 mL, about 900 mL, about 1 L, about2 L, about 3 L, about 4 L, about 5 L, about 6 L, about 7 L, about 8 L,about 9 L, and about 10 L.

In an embodiment, TILs can be expanded in G-Rex flasks (commerciallyavailable from Wilson Wolf Manufacturing). Such embodiments allow forcell populations to expand from about 5×10⁵ cells/cm′ to between 10×10⁶and 30×10⁶ cells/cm′. In an embodiment this is without feeding. In anembodiment, this is without feeding so long as medium resides at aheight of about 10 cm in the G-Rex flask. In an embodiment this iswithout feeding but with the addition of one or more cytokines. In anembodiment, the cytokine can be added as a bolus without any need to mixthe cytokine with the medium. Such containers, devices, and methods areknown in the art and have been used to expand TILs, and include thosedescribed in U.S. Patent Application Publication No. US 2014/0377739A1,International Publication No. WO 2014/210036 A1, U.S. Patent ApplicationPublication No. us 2013/0115617 A1, International Publication No. WO2013/188427 A1, U.S. Patent Application Publication No. US 2011/0136228A1, U.S. Pat. No. 8,809,050 B2, International publication No. WO2011/072088 A2, U.S. Patent Application Publication No. US 2016/0208216A1, U.S. Patent Application Publication No. US 2012/0244133 A1,International Publication No. WO 2012/129201 A1, U.S. Patent ApplicationPublication No. US 2013/0102075 A1, U.S. Pat. No. 8,956,860 B2,International Publication No. WO 2013/173835 A1, U.S. Patent ApplicationPublication No. US 2015/0175966 A1, the disclosures of which areincorporated herein by reference. Such processes are also described inJin et al., J. Immunotherapy, 2012, 35:283-292.

Optional Genetic Engineering of TILs

In some embodiments, the TILs are optionally genetically engineered toinclude additional functionalities, including, but not limited to, ahigh-affinity T cell receptor (TCR), e.g., a TCR targeted at atumor-associated antigen such as MAGE-1, HER2, or NY-ESO-1, or achimeric antigen receptor (CAR) which binds to a tumor-associated cellsurface molecule (e.g., mesothelin) or lineage-restricted cell surfacemolecule (e.g., CD19).

Optional Cryopreservation of TILs

As discussed above, and exemplified in Steps A through E as provided inFIG. 18, cryopreservation can occur at numerous points throughout theTIL expansion process. In some embodiments, the expanded population ofTILs after the second expansion (as provided for example, according toStep D of FIG. 18) can be cryopreserved. Cryopreservation can begenerally accomplished by placing the TIL population into a freezingsolution, e.g., 85% complement inactivated AB serum and 15% dimethylsulfoxide (DMSO). The cells in solution are placed into cryogenic vialsand stored for 24 hours at −80° C., with optional transfer to gaseousnitrogen freezers for cryopreservation. See Sadeghi, et al., ActaOncologica 2013, 52, 978-986. In some embodiments, the TILs arecryopreserved in 5% DMSO. In some embodiments, the TILs arecryopreserved in cell culture media plus 5% DMSO. In some embodiments,the TILs are cryopreserved according to the methods provided in Example10.

When appropriate, the cells are removed from the freezer and thawed in a37° C. water bath until approximately ⅘ of the solution is thawed. Thecells are generally resuspended in complete media and optionally washedone or more times. In some embodiments, the thawed TILs can be countedand assessed for viability as is known in the art.

Closed Systems for TIL Manufacturing

The present invention provides for the use of closed systems during theTIL culturing process. Such closed systems allow for preventing and/orreducing microbial contamination, allow for the use of fewer flasks, andallow for cost reductions. In some embodiments, the closed system usestwo containers.

Such closed systems are well-known in the art and can be found, forexample, at http://www.fda.gov/cber/guidelines.htm andhttps://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guidances/Blood/ucm076779.htm.

Sterile connecting devices (STCDs) produce sterile welds between twopieces of compatible tubing. This procedure permits sterile connectionof a variety of containers and tube diameters. In some embodiments, theclosed systems include luer lock and heat sealed systems. In someembodiments, the closed system is accessed via syringes under sterileconditions in order to maintain the sterility and closed nature of thesystem. In some embodiments, a closed system as described in theExamples is employed. In some embodiments, the TILs are formulated intoa final product formulation container according to the method describedin the Examples.

In some embodiments, the closed system uses one container from the timethe tumor fragments are obtained until the TILs are ready foradministration to the patient or cryopreserving. In some embodimentswhen two containers are used, the first container is a closedG-container and the population of TILs is centrifuged and transferred toan infusion bag without opening the first closed G-container. In someembodiments, when two containers are used, the infusion bag is aHypoThermosol-containing infusion bag. A closed system or closed TILcell culture system is characterized in that once the tumor sampleand/or tumor fragments have been added, the system is tightly sealedfrom the outside to form a closed environment free from the invasion ofbacteria, fungi, and/or any other microbial contamination.

In some embodiments, the reduction in microbial contamination is betweenabout 5% and about 100%. In some embodiments, the reduction in microbialcontamination is between about 5% and about 95%. In some embodiments,the reduction in microbial contamination is between about 5% and about90%. In some embodiments, the reduction in microbial contamination isbetween about 10% and about 90%. In some embodiments, the reduction inmicrobial contamination is between about 15% and about 85%. In someembodiments, the reduction in microbial contamination is about 5%, about10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%,about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%,about 99%, or about 100%.

The closed system allows for TIL growth in the absence and/or with asignificant reduction in microbial contamination.

Moreover, pH, carbon dioxide partial pressure and oxygen partialpressure of the TIL cell culture environment each vary as the cells arecultured. Consequently, even though a medium appropriate for cellculture is circulated, the closed environment still needs to beconstantly maintained as an optimal environment for TIL proliferation.To this end, it is desirable that the physical factors of pH, carbondioxide partial pressure and oxygen partial pressure within the cultureliquid of the closed environment be monitored by means of a sensor, thesignal whereof is used to control a gas exchanger installed at the inletof the culture environment, and the that gas partial pressure of theclosed environment be adjusted in real time according to changes in theculture liquid so as to optimize the cell culture environment. In someembodiments, the present invention provides a closed cell culture systemwhich incorporates at the inlet to the closed environment a gasexchanger equipped with a monitoring device which measures the pH,carbon dioxide partial pressure and oxygen partial pressure of theclosed environment, and optimizes the cell culture environment byautomatically adjusting gas concentrations based on signals from themonitoring device.

In some embodiments, the pressure within the closed environment iscontinuously or intermittently controlled. That is, the pressure in theclosed environment can be varied by means of a pressure maintenancedevice for example, thus ensuring that the space is suitable for growthof TILs in a positive pressure state, or promoting exudation of fluid ina negative pressure state and thus promoting cell proliferation. Byapplying negative pressure intermittently, moreover, it is possible touniformly and efficiently replace the circulating liquid in the closedenvironment by means of a temporary shrinkage in the volume of theclosed environment.

In some embodiments, optimal culture components for proliferation of theTILs can be substituted or added, and including factors such as IL-2and/or OKT3, as well as combination, can be added.

Optional Cryopreservation of TILs

Either the bulk TIL population or the expanded population of TILs can beoptionally cryopreserved. In some embodiments, cryopreservation occurson the therapeutic TIL population. In some embodiments, cryopreservationoccurs on the TILs harvested after the second expansion. In someembodiments, cryopreservation occurs on the TILs in exemplary Step F ofFIG. 18. In some embodiments, the TILs are cryopreserved in the infusionbag. In some embodiments, the TILs are cryopreserved prior to placementin an infusion bag. In some embodiments, the TILs are cryopreserved andnot placed in an infusion bag. In some embodiments, cryopreservation isperformed using a cryopreservation medium. In some embodiments, thecryopreservation media contains dimethylsulfoxide (DMSO). This isgenerally accomplished by putting the TIL population into a freezingsolution, e.g. 85% complement inactivated AB serum and 15% dimethylsulfoxide (DMSO). The cells in solution are placed into cryogenic vialsand stored for 24 hours at −80° C., with optional transfer to gaseousnitrogen freezers for cryopreservation. See, Sadeghi, et al., ActaOncologica 2013, 52, 978-986.

When appropriate, the cells are removed from the freezer and thawed in a37° C. water bath until approximately ⅘ of the solution is thawed. Thecells are generally resuspended in complete media and optionally washedone or more times. In some embodiments, the thawed TILs can be countedand assessed for viability as is known in the art.

In a preferred embodiment, a population of TILs is cryopreserved usingCS10 cryopreservation media (CryoStor 10, BioLife Solutions). In apreferred embodiment, a population of TILs is cryopreserved using acryopreservation media containing dimethylsulfoxide (DMSO). In apreferred embodiment, a population of TILs is cryopreserved using a 1:1(vol:vol) ratio of CS10 and cell culture media. In a preferredembodiment, a population of TILs is cryopreserved using about a 1:1(vol:vol) ratio of CS10 and cell culture media, further comprisingadditional IL-2.

As discussed above in Steps A through E, cryopreservation can occur atnumerous points throughout the TIL expansion process. In someembodiments, the bulk TIL population after the first expansion accordingto Step B or the expanded population of TILs after the one or moresecond expansions according to Step D can be cryopreserved.Cryopreservation can be generally accomplished by placing the TILpopulation into a freezing solution, e.g., 85% complement inactivated ABserum and 15% dimethyl sulfoxide (DMSO). The cells in solution areplaced into cryogenic vials and stored for 24 hours at −80° C., withoptional transfer to gaseous nitrogen freezers for cryopreservation. SeeSadeghi, et al., Acta Oncologica 2013, 52, 978-986.

When appropriate, the cells are removed from the freezer and thawed in a37° C. water bath until approximately ⅘ of the solution is thawed. Thecells are generally resuspended in complete media and optionally washedone or more times. In some embodiments, the thawed TILs can be countedand assessed for viability as is known in the art.

In some cases, the Step B TIL population can be cryopreservedimmediately, using the protocols discussed below. Alternatively, thebulk TIL population can be subjected to Step C and Step D and thencryopreserved after Step D. Similarly, in the case where geneticallymodified TILs will be used in therapy, the Step B or Step D TILpopulations can be subjected to genetic modifications for suitabletreatments.

Pharmaceutical Compositions, Dosages, and Dosing Regimens

In an embodiment, TILs expanded using the methods of the presentdisclosure are administered to a patient as a pharmaceuticalcomposition. In an embodiment, the pharmaceutical composition is asuspension of TILs in a sterile buffer. TILs expanded using PBMCs of thepresent disclosure may be administered by any suitable route as known inthe art. In some embodiments, the T-cells are administered as a singleintra-arterial or intravenous infusion, which preferably lastsapproximately 30 to 60 minutes. Other suitable routes of administrationinclude intraperitoneal, intrathecal, and intralymphatic administration.

Any suitable dose of TILs can be administered. In some embodiments, fromabout 2.3×10¹⁰ to about 13.7×10¹⁰ TILs are administered, with an averageof around 7.8×10¹⁰ TILs, particularly if the cancer is melanoma. In anembodiment, about 1.2×10¹⁰ to about 4.3×10¹⁰ of TILs are administered.In some embodiments, about 3×10¹⁰ to about 12×10¹⁰ TILs areadministered. In some embodiments, about 4×10¹⁰ to about 10×10¹⁰ TILsare administered. In some embodiments, about 5×10¹⁰ to about 8×10¹⁰ TILsare administered. In some embodiments, about 6×10¹⁰ to about 8×10¹⁰ TILsare administered. In some embodiments, about 7×10¹⁰ to about 8×10¹⁰ TILsare administered. In some embodiments, the therapeutically effectivedosage is about 2.3×10¹⁰ to about 13.7×10¹⁰. In some embodiments, thetherapeutically effective dosage is about 7.8×10¹⁰ TILs, particularly ofthe cancer is melanoma. In some embodiments, the therapeuticallyeffective dosage is about 1.2×10¹⁰ to about 4.3×10¹⁰ of TILs. In someembodiments, the therapeutically effective dosage is about 3×10¹⁰ toabout 12×10¹⁰ TILs. In some embodiments, the therapeutically effectivedosage is about 4×10¹⁰ to about 10×10¹⁰ TILs. In some embodiments, thetherapeutically effective dosage is about 5×10¹⁰ to about 8×10¹⁰ TILs.In some embodiments, the therapeutically effective dosage is about6×10¹⁰ to about 8×10¹⁰ TILs. In some embodiments, the therapeuticallyeffective dosage is about 7×10¹⁰ to about 8×10¹⁰ TILs.

In some embodiments, the number of the TILs provided in thepharmaceutical compositions of the invention is about 1×10⁶, 2×10⁶,3×10⁶, 4×10⁶, 5×10⁶, 6×10⁶, 7×10⁶, 8×10⁶, 9×10⁶, 1×10⁷, 2×10⁷, 3×10⁷,4×10⁷, 5×10⁷, 6×10⁷, 7×10⁷, 8×10⁷, 9×10⁷, 1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸,5×10⁸, 6×10⁸, 7×10⁸, 8×10⁸, 9×10⁸, 1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹,6×10⁹, 7×10⁹, 8×10⁹, 9×10⁹, 1×10¹⁰, 2×10¹⁰, 3×10¹⁰, 4×10¹⁰, 5×10¹⁰,6×10¹⁰, 7×10¹⁰, 8×10¹⁰, 9×10¹⁰, 1×10¹¹, 2×10¹¹, 3×10¹¹, 4×10¹¹, 5×10¹¹,6×10¹¹, 7×10¹¹, 8×10¹¹, 9×10¹¹, 1×10¹², 2×10¹², 3×10¹², 4×10¹², 5×10¹²,6×10¹², 7×10¹² 8×10¹², 9×10¹², 1×10¹³, 2×10¹³, 3×10¹³ 4×10¹³, 5×10¹³6×10¹³, 7×10¹³, 8×10¹³, and 9×10¹³. In an embodiment, the number of theTILs provided in the pharmaceutical compositions of the invention is inthe range of 1×10⁶ to 5×10⁶, 5×10⁶ to 1×10⁷, 1×10⁷ to 5×10⁷, 5×10⁷ to1×10⁸, 1×10⁸ to 5×10⁸, 5×10⁸ to 1×10⁹, 1×10⁹ to 5×10⁹, 5×10⁹ to 1×10¹⁰,1×10¹⁰ to 5×10¹⁰, 5×10¹⁰ to 1×10¹¹, 5×10¹¹ to 1×10¹², 1×10¹² to 5×10¹²,and 5×10¹² to 1×10¹³.

In some embodiments, the concentration of the TILs provided in thepharmaceutical compositions of the invention is less than, for example,100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%,14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%,0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%,0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%,0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%,0.0003%, 0.0002% or 0.0001% w/w, w/v or v/v of the pharmaceuticalcomposition.

In some embodiments, the concentration of the TILs provided in thepharmaceutical compositions of the invention is greater than 90%, 80%,70%, 60%, 50%, 40%, 30%, 20%, 19.75%, 19.50%, 19.25% 19%, 18.75%,18.50%, 18.25% 18%, 17.75%, 17.50%, 17.25% 17%, 16.75%, 16.50%, 16.25%16%, 15.75%, 15.50%, 15.25% 15%, 14.75%, 14.50%, 14.25% 14%, 13.75%,13.50%, 13.25% 13%, 12.75%, 12.50%, 12.25% 12%, 11.75%, 11.50%, 11.25%11%, 10.75%, 10.50%, 10.25% 10%, 9.75%, 9.50%, 9.25% 9%, 8.75%, 8.50%,8.25% 8%, 7.75%, 7.50%, 7.25% 7%, 6.75%, 6.50%, 6.25% 6%, 5.75%, 5.50%,5.25% 5%, 4.75%, 4.50%, 4.25%, 4%, 3.75%, 3.50%, 3.25%, 3%, 2.75%,2.50%, 2.25%, 2%, 1.75%, 1.50%, 125%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%,0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%,0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%,0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001%w/w, w/v, or v/v of the pharmaceutical composition.

In some embodiments, the concentration of the TILs provided in thepharmaceutical compositions of the invention is in the range from about0.0001% to about 50%, about 0.001% to about 40%, about 0.01% to about30%, about 0.02% to about 29%, about 0.03% to about 28%, about 0.04% toabout 27%, about 0.05% to about 26%, about 0.06% to about 25%, about0.07% to about 24%, about 0.08% to about 23%, about 0.09% to about 22%,about 0.1% to about 21%, about 0.2% to about 20%, about 0.3% to about19%, about 0.4% to about 18%, about 0.5% to about 17%, about 0.6% toabout 16%, about 0.7% to about 15%, about 0.8% to about 14%, about 0.9%to about 12% or about 1% to about 10% w/w, w/v or v/v of thepharmaceutical composition.

In some embodiments, the concentration of the TILs provided in thepharmaceutical compositions of the invention is in the range from about0.001% to about 10%, about 0.01% to about 5%, about 0.02% to about 4.5%,about 0.03% to about 4%, about 0.04% to about 3.5%, about 0.05% to about3%, about 0.06% to about 2.5%, about 0.07% to about 2%, about 0.08% toabout 1.5%, about 0.09% to about 1%, about 0.1% to about 0.9% w/w, w/vor v/v of the pharmaceutical composition.

In some embodiments, the amount of the TILs provided in thepharmaceutical compositions of the invention is equal to or less than 10g, 9.5 g, 9.0 g, 8.5 g, 8.0 g, 7.5 g, 7.0 g, 6.5 g, 6.0 g, 5.5 g, 5.0 g,4.5 g, 4.0 g, 3.5 g, 3.0 g, 2.5 g, 2.0 g, 1.5 g, 1.0 g, 0.95 g, 0.9 g,0.85 g, 0.8 g, 0.75 g, 0.7 g, 0.65 g, 0.6 g, 0.55 g, 0.5 g, 0.45 g, 0.4g, 0.35 g, 0.3 g, 0.25 g, 0.2 g, 0.15 g, 0.1 g, 0.09 g, 0.08 g, 0.07 g,0.06 g, 0.05 g, 0.04 g, 0.03 g, 0.02 g, 0.01 g, 0.009 g, 0.008 g, 0.007g, 0.006 g, 0.005 g, 0.004 g, 0.003 g, 0.002 g, 0.001 g, 0.0009 g,0.0008 g, 0.0007 g, 0.0006 g, 0.0005 g, 0.0004 g, 0.0003 g, 0.0002 g, or0.0001 g.

In some embodiments, the amount of the TILs provided in thepharmaceutical compositions of the invention is more than 0.0001 g,0.0002 g, 0.0003 g, 0.0004 g, 0.0005 g, 0.0006 g, 0.0007 g, 0.0008 g,0.0009 g, 0.001 g, 0.0015 g, 0.002 g, 0.0025 g, 0.003 g, 0.0035 g, 0.004g, 0.0045 g, 0.005 g, 0.0055 g, 0.006 g, 0.0065 g, 0.007 g, 0.0075 g,0.008 g, 0.0085 g, 0.009 g, 0.0095 g, 0.01 g, 0.015 g, 0.02 g, 0.025 g,0.03 g, 0.035 g, 0.04 g, 0.045 g, 0.05 g, 0.055 g, 0.06 g, 0.065 g, 0.07g, 0.075 g, 0.08 g, 0.085 g, 0.09 g, 0.095 g, 0.1 g, 0.15 g, 0.2 g, 0.25g, 0.3 g, 0.35 g, 0.4 g, 0.45 g, 0.5 g, 0.55 g, 0.6 g, 0.65 g, 0.7 g,0.75 g, 0.8 g, 0.85 g, 0.9 g, 0.95 g, 1 g, 1.5 g, 2 g, 2.5, 3 g, 3.5, 4g, 4.5 g, 5 g, 5.5 g, 6 g, 6.5 g, 7 g, 7.5 g, 8 g, 8.5 g, 9 g, 9.5 g, or10 g.

The TILs provided in the pharmaceutical compositions of the inventionare effective over a wide dosage range. The exact dosage will dependupon the route of administration, the form in which the compound isadministered, the gender and age of the subject to be treated, the bodyweight of the subject to be treated, and the preference and experienceof the attending physician. The clinically-established dosages of theTILs may also be used if appropriate. The amounts of the pharmaceuticalcompositions administered using the methods herein, such as the dosagesof TILs, will be dependent on the human or mammal being treated, theseverity of the disorder or condition, the rate of administration, thedisposition of the active pharmaceutical ingredients and the discretionof the prescribing physician.

In some embodiments, TILs may be administered in a single dose. Suchadministration may be by injection, e.g., intravenous injection. In someembodiments, TILs may be administered in multiple doses. Dosing may beonce, twice, three times, four times, five times, six times, or morethan six times per year. Dosing may be once a month, once every twoweeks, once a week, or once every other day. Administration of TILs maycontinue as long as necessary.

In some embodiments, an effective dosage of TILs is about 1×10⁶, 2×10⁶,3×10⁶, 4×10⁶, 5×10⁶, 6×10⁶, 7×10⁶, 8×10⁶, 9×10⁶, 1×10⁷, 2×10⁷, 3×10⁷,4×10⁷, 5×10⁷, 6×10⁷, 7×10⁷, 8×10⁷, 9×10⁷, 1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸,5×10⁸, 6×10⁸, 7×10⁸, 8×10⁸, 9×10⁸, 1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹,6×10⁹, 7×10⁹, 8×10⁹, 9×10⁹, 1×10¹⁰, 2×10¹⁰, 3×10¹⁰, 4×10¹⁰, 5×10¹⁰,6×10¹⁰, 7×10¹⁰, 8×10¹⁰, 9×10¹⁰, 1×10¹¹, 2×10¹¹, 3×10¹¹, 4×10¹¹, 5×10¹¹,6×10¹¹, 7×10¹¹, 8×10¹¹, 9×10¹¹, 1×10¹², 2×10¹², 3×10¹², 4×10¹², 5×10¹²,6×10¹², 7×10¹², 8×10¹², 9×10¹², 1×10¹³, 2×10¹³ 3×10¹³, 4×10¹³, 5×10¹³,6×10¹³, 7×10¹³, 8×10¹³, and 9×10¹³. In some embodiments, an effectivedosage of TILs is in the range of 1×10⁶ to 5×10⁶, 5×10⁶ to 1×10⁷, 1×10to 5×10⁷, 5×10⁷ to 1×10⁸, 1×10⁸ to 5×10⁸, 5×10⁸ to 1×10⁹, 1×10⁹ to5×10⁹, 5×10⁹ to 1×10¹⁰, 1×10¹⁰ to 5×10¹⁰ 5×10¹⁰ to 1×10¹¹, 5×10¹¹ to1×10¹², 1×10¹² to 5×10¹², and 5×10¹² to 1×10¹³.

In some embodiments, an effective dosage of TILs is in the range ofabout 0.01 mg/kg to about 4.3 mg/kg, about 0.15 mg/kg to about 3.6mg/kg, about 0.3 mg/kg to about 3.2 mg/kg, about 0.35 mg/kg to about2.85 mg/kg, about 0.15 mg/kg to about 2.85 mg/kg, about 0.3 mg to about2.15 mg/kg, about 0.45 mg/kg to about 1.7 mg/kg, about 0.15 mg/kg toabout 1.3 mg/kg, about 0.3 mg/kg to about 1.15 mg/kg, about 0.45 mg/kgto about 1 mg/kg, about 0.55 mg/kg to about 0.85 mg/kg, about 0.65 mg/kgto about 0.8 mg/kg, about 0.7 mg/kg to about 0.75 mg/kg, about 0.7 mg/kgto about 2.15 mg/kg, about 0.85 mg/kg to about 2 mg/kg, about 1 mg/kg toabout 1.85 mg/kg, about 1.15 mg/kg to about 1.7 mg/kg, about 1.3 mg/kgmg to about 1.6 mg/kg, about 1.35 mg/kg to about 1.5 mg/kg, about 2.15mg/kg to about 3.6 mg/kg, about 2.3 mg/kg to about 3.4 mg/kg, about 2.4mg/kg to about 3.3 mg/kg, about 2.6 mg/kg to about 3.15 mg/kg, about 2.7mg/kg to about 3 mg/kg, about 2.8 mg/kg to about 3 mg/kg, or about 2.85mg/kg to about 2.95 mg/kg.

In some embodiments, an effective dosage of TILs is in the range ofabout 1 mg to about 500 mg, about 10 mg to about 300 mg, about 20 mg toabout 250 mg, about 25 mg to about 200 mg, about 1 mg to about 50 mg,about 5 mg to about 45 mg, about 10 mg to about 40 mg, about 15 mg toabout 35 mg, about 20 mg to about 30 mg, about 23 mg to about 28 mg,about 50 mg to about 150 mg, about 60 mg to about 140 mg, about 70 mg toabout 130 mg, about 80 mg to about 120 mg, about 90 mg to about 110 mg,or about 95 mg to about 105 mg, about 98 mg to about 102 mg, about 150mg to about 250 mg, about 160 mg to about 240 mg, about 170 mg to about230 mg, about 180 mg to about 220 mg, about 190 mg to about 210 mg,about 195 mg to about 205 mg, or about 198 to about 207 mg.

An effective amount of the TILs may be administered in either single ormultiple doses by any of the accepted modes of administration of agentshaving similar utilities, including intranasal and transdermal routes,by intra-arterial injection, intravenously, intraperitoneally,parenterally, intramuscularly, subcutaneously, topically, bytransplantation, or by inhalation.

Biomarker Correlation for Predicting Treatment Efficacy and/or Response

Interferon gamma inducible protein 10 (IP-1) (also known as CXCL10) is a10 kDa chemokine that was originally identified based on expression ofthe IP-10 gene in cells treated with interferon gamma (IFN-gamma)(Luster, A. D. et al. (1985) Nature 315:672-676) The sequence for humanIP-10 can be found in as Genbank Acc. No. NP_001556. The presentinvention provides methods of predicting treatment response and/ortherapeutic efficacy of a TIL therapy administered as described herein.

In some aspects of the invention, IP-10 is measured after treatment of apatient with a therapeutic population of tumor infiltrating lymphocytes(TILs). In some embodiments, any method know in the art for detectingIP-10 can be employed. For example, some methods for detection includethe use of IP antibodies, including for example, those described in U.S.Patent Publication No. 2005/0191293. In some embodiments, the level ofIP-10 is the level of IP-10 protein in a sample. In some embodiments,IP-10 is measured by a commercial Bio-Rad bead-based Bio-Pleximmunoassay, which measures multiple cytokines and chemokines, and whichincludes an antibody specific for IP-10. In some embodiments, IP-10 ismeasured by taking blood samples from the patient and is measured in theplasma fraction obtained from the blood (i.e., after all blood cells areremoved) and is reported in units of picograms per milliliter of plasma.In some embodiments, the level of IP-10 is measured by calculating thedifference between the level of IP-10 before administration of thetherapeutic population of TILs and the level of IP-10 afteradministration of the therapeutic population of TILs. In someembodiments, the level of IP-10 is measured by calculating thedifference between the level of IP-10 at Day −7 before administration ofthe therapeutic population of TILs and the level of IP-10 at Day 1 afteradministration of the therapeutic population of TILs, wherein Day 0 isthe Day of the TIL infusion/administration.

The present invention provides methods of treating cancer, includingtreating double-refractory metastatic melanoma, in a patient in need byadministering a therapeutically effective population of tumorinfiltrating lymphocytes (TILs) to the patient, where the patient isshown to exhibit an increase in IP-10 after administration of thetherapeutically effective population of tumor infiltrating lymphocytes(TILs). In some embodiments, and the increase in the level of IP-10 isindicative of treatment response and/or treatment efficacy of the TILadministration. In some embodiments, the increase in the level of IP-10is at least one-fold to at least five-fold, as compared to the level ofIP-10 in the patient before the TIL administration. In some embodiments,the IP-10 is measured before another treatment regimen, such as ananti-PD-1 treatment regimen. In some embodiments, the IP-10 is measuredafter another treatment regimen, such as an anti-PD-1 treatment regimen,but before administration of TILs. In some embodiments, the level ofIP-10 is measured before the TILs are harvested for expansion. In someembodiments, the level of IP-10 is measured after the TILs are harvestedfor expansion. In some embodiments, the level IP-10 is measured 6 hoursto 24 hours prior to administration of the TILs to the patient. In someembodiments, the level of IP-10 is measured 1 day, 2 days, 3 days, 4days, 5 days or more before administration of the TILs to the patient.In some embodiments, the level of IP-10 is measured by calculating thedifference between the level of IP-10 before administration of thetherapeutic population of TILs and the level of IP-10 afteradministration of the therapeutic population of TILs. In someembodiments, the level of IP-10 is measured by calculating thedifference between the level of IP-10 at Day −7 before administration ofthe therapeutic population of TILs and the level of IP-10 at Day 1 afteradministration of the therapeutic population of TILs, wherein Day 0 isthe Day of the TIL infusion/administration. In some embodiments, thethreshold value for IP-10 level is about 500 pg/ml to about 3500 pg/mL,wherein an IP-10 level above the threshold value is indicative oftreatment efficacy and/or treatment response. In some embodiments, thethreshold value for IP-10 level is at least 1000 pg/mL, wherein an IP-10level above the threshold value is indicative of treatment efficacyand/or treatment response. In some embodiments, the threshold value forIP-10 level is at least 1500 pg/mL, wherein an IP-10 level above thethreshold value is indicative of treatment efficacy and/or treatmentresponse. In some embodiments, the threshold value for IP-10 level is atleast 2000 pg/mL, wherein an IP-10 level above the threshold value isindicative of treatment efficacy and/or treatment response. In someembodiments, the threshold value for IP-10 level is at least 2500 pg/mL,wherein an IP-10 level above the threshold value is indicative oftreatment efficacy and/or treatment response. In some embodiments, thelevel of IP-10 is about 1000 pg/ml to about 3000 pg/mL, wherein an IP-10level above the threshold value is indicative of treatment efficacyand/or treatment response. In some embodiments, the level of IP-10 is atleast 1000 pg/mL and the IP-10 level is indicative of treatment efficacyand/or treatment response. In some embodiments, the level of IP-10 is atleast 1500 pg/mL and the IP-10 level is indicative of treatment efficacyand/or treatment response. In some embodiments, the level of IP-10 is atleast 2000 pg/mL and the IP-10 level is indicative of treatment efficacyand/or treatment response. In some embodiments, the level of IP-10 is atleast 2500 pg/mL and the IP-10 level is indicative of treatment efficacyand/or treatment response. In some embodiments, the increase in thelevel of IP-10 is measured by calculating the difference in IP-10 levelin plasma seven days before TIL infusion and one day after TIL infusion,and wherein said difference in IP-10 level in plasma is at least 800pg/mL, at least 900 pg/mL, at least 1000 pg/mL, at least 1100 pg/mL, atleast 1200 pg/mL, at least 1300 pg/mL, at least 1400 pg/mL, at least1500 pg/mL, at least 1600 pg/mL, at least 1700 pg/mL, or at least 1800pg/mL, at least 1900 pg/mL, at least 2000 pg/mL, at least 2100 pg/mL, orat least 2200 pg/mL. In some embodiments, when there is an increase inthe level of IP-10 after administration of a therapeutically effectivepopulation of tumor infiltrating lymphocytes, the patient isadministered one or more further dosages of a therapeutically effectivepopulation of tumor infiltrating lymphocytes (TILs). In someembodiments, when there is an increase in the level of IP-10 afteradministration of a therapeutically effective population of tumorinfiltrating lymphocytes, patient is not administered a further dosageof a therapeutically effective population of tumor infiltratinglymphocytes (TILs). In some embodiments, the therapeutically effectivepopulation that results in an increase in the level of IP-10 afteradministration of TILs comprises from about 2.3×10¹⁰ to about 13.7×10¹⁰TILs. In some embodiments, the one, two, three or more therapeutic TILdosages are administered after determining there is an increase in thelevel of IP-10.

In some embodiments the invention provides methods of treating cancer ordouble-refractory metastatic melanoma in a patient where the methodcomprises: (a) obtaining a first population of TILs from a tumorresected from the patient by processing a tumor sample obtained from thepatient into multiple tumor fragments; (b) adding the tumor fragmentsinto a closed system; (c) performing a first expansion by culturing thefirst population of TILs in a cell culture medium comprising IL-2, andoptionally OKT-3, to produce a second population of TILs, wherein thefirst expansion is performed in a closed container providing a firstgas-permeable surface area, wherein the first expansion is performed forabout 3-14 days to obtain the second population of TILs, wherein thesecond population of TILs is at least 50-fold greater in number than thefirst population of TILs, and wherein the transition from step (b) tostep (c) occurs without opening the system; (d) performing a secondexpansion by supplementing the cell culture medium of the secondpopulation of TILs with additional IL-2, optionally OKT-3, and antigenpresenting cells (APCs), to produce a third population of TILs, whereinthe second expansion is performed for about 7-14 days to obtain thethird population of TILs, wherein the third population of TILs is atherapeutic population of TILs, wherein the second expansion isperformed in a closed container providing a second gas-permeable surfacearea, and wherein the transition from step (c) to step (d) occurswithout opening the system; (e) harvesting the therapeutic population ofTILs obtained from step (d) to provide a harvested TIL population,wherein the transition from step (d) to step (e) occurs without openingthe system; (f) transferring the harvested TIL population from step (e)to an infusion bag, wherein the transfer from step (e) to (f) occurswithout opening the system, and optionally cryopreserving the harvestedTIL population and (g) administering a therapeutically effective amountof the harvested TIL population to the patient with cancer ordouble-refractory metastatic melanoma. In some embodiments, the patientexhibits an increase in IP-10 after administration of thetherapeutically effective population of tumor infiltrating lymphocytes(TILs). In some embodiments, the patient is shown to exhibit an increasein IP-10 after administration of the therapeutically effectivepopulation of tumor infiltrating lymphocytes (TILs). In someembodiments, and the increase in the level of IP-10 is indicative oftreatment response and/or treatment efficacy of the TIL administration.In some embodiments, the increase in the level of IP-10 is at leastone-fold to at least five-fold, as compared to the level of IP-10 in thepatient before the TIL administration. In some embodiments, the IP-10 ismeasured before another treatment regimen, such as an anti-PD-1treatment regimen. In some embodiments, the IP-10 is measured afteranother treatment regimen, such as an anti-PD-1 treatment regimen, butbefore administration of TILs. In some embodiments, the level of IP-10is measured before the TILs are harvested for expansion. In someembodiments, the level of IP-10 is measured after the TILs are harvestedfor expansion. In some embodiments, the level IP-10 is measured 6 hoursto 24 hours prior to administration of the TILs to the patient. In someembodiments, the level of IP-10 is measured 1 day, 2 days, 3 days, 4days, 5 days or more before administration of the TILs to the patient.In some embodiments, when there is an increase in the level of IP-10after administration of a therapeutically effective population of tumorinfiltrating lymphocytes, the patient is administered one or morefurther dosages of a therapeutically effective population of tumorinfiltrating lymphocytes (TILs). In some embodiments, when there is anincrease in the level of IP-10 after administration of a therapeuticallyeffective population of tumor infiltrating lymphocytes, patient is notadministered a further dosage of a therapeutically effective populationof tumor infiltrating lymphocytes (TILs). In some embodiments, thetherapeutically effective population that results in an increase in thelevel of IP-10 after administration of TILs comprises from about2.3×10¹⁰ to about 13.7×10¹⁰ TILs. In some embodiments, the one, two,three or more therapeutic TIL dosages are administered after determiningthere is an increase in the level of IP-10. In some embodiments, thelevel of IP-10 is measured by calculating the difference between thelevel of IP-10 before administration of the therapeutic population ofTILs and the level of IP-10 after administration of the therapeuticpopulation of TILs. In some embodiments, the level of IP-10 is measuredby calculating the difference between the level of IP-10 at Day −7before administration of the therapeutic population of TILs and thelevel of IP-10 at Day 1 after administration of the therapeuticpopulation of TILs, wherein Day 0 is the Day of the TILinfusion/administration. In some embodiments, the threshold value forIP-10 level is about 500 pg/ml to about 3500 pg/mL, wherein an IP-10level above the threshold value is indicative of treatment efficacyand/or treatment response. In some embodiments, the threshold value forIP-10 level is at least 1000 pg/mL, wherein an IP-10 level above thethreshold value is indicative of treatment efficacy and/or treatmentresponse. In some embodiments, the threshold value for IP-10 level is atleast 1500 pg/mL, wherein an IP-10 level above the threshold value isindicative of treatment efficacy and/or treatment response. In someembodiments, the threshold value for IP-10 level is at least 2000 pg/mL,wherein an IP-10 level above the threshold value is indicative oftreatment efficacy and/or treatment response. In some embodiments, thethreshold value for IP-10 level is at least 2500 pg/mL, wherein an IP-10level above the threshold value is indicative of treatment efficacyand/or treatment response. In some embodiments, the level of IP-10 isabout 1000 pg/ml to about 3000 pg/mL, wherein an IP-10 level above thethreshold value is indicative of treatment efficacy and/or treatmentresponse. In some embodiments, the level of IP-10 is at least 1000 pg/mLand the IP-10 level is indicative of treatment efficacy and/or treatmentresponse. In some embodiments, the level of IP-10 is at least 1500 pg/mLand the IP-10 level is indicative of treatment efficacy and/or treatmentresponse. In some embodiments, the level of IP-10 is at least 2000 pg/mLand the IP-10 level is indicative of treatment efficacy and/or treatmentresponse. In some embodiments, the level of IP-10 is at least 2500 pg/mLand the IP-10 level is indicative of treatment efficacy and/or treatmentresponse. In some embodiments, the increase in the level of IP-10 ismeasured by calculating the difference in IP-10 level in plasma sevendays before TIL infusion and one day after TIL infusion, and whereinsaid difference in IP-10 level in plasma is at least 800 pg/mL, at least900 pg/mL, at least 1000 pg/mL, at least 1100 pg/mL, at least 1200pg/mL, at least 1300 pg/mL, at least 1400 pg/mL, at least 1500 pg/mL, atleast 1600 pg/mL, at least 1700 pg/mL, or at least 1800 pg/mL, at least1900 pg/mL, at least 2000 pg/mL, at least 2100 pg/mL, or at least 2200pg/mL.

In some embodiments the invention provides methods of treating cancer ordouble-refractory metastatic melanoma in a patient in need thereof, themethod comprising: (a) obtaining a first population of TILs from a tumorresected from the patient by processing a tumor sample obtained from thepatient into multiple tumor fragments; (b) adding the tumor fragmentsinto a closed system; (c) performing a first expansion by culturing thefirst population of TILs in a cell culture medium comprising IL-2, andoptionally OKT-3, to produce a second population of TILs, wherein thefirst expansion is performed in a closed container providing a firstgas-permeable surface area, wherein the first expansion is performed forabout 3-14 days to obtain the second population of TILs, wherein thesecond population of TILs is at least 50-fold greater in number than thefirst population of TILs, and wherein the transition from step (b) tostep (c) occurs without opening the system; (d) performing a secondexpansion by supplementing the cell culture medium of the secondpopulation of TILs with additional IL-2, optionally OKT-3, and antigenpresenting cells (APCs), to produce a third population of TILs, whereinthe second expansion is performed for about 7-14 days to obtain thethird population of TILs, wherein the third population of TILs is atherapeutic population of TILs, wherein the second expansion isperformed in a closed container providing a second gas-permeable surfacearea, and wherein the transition from step (c) to step (d) occurswithout opening the system; (e) harvesting the therapeutic population ofTILs obtained from step (d) to provide a harvested TIL population,wherein the transition from step (d) to step (e) occurs without openingthe system; (f) transferring the harvested TIL population from step (e)to an infusion bag, wherein the transfer from step (e) to (f) occurswithout opening the system, and optionally cryopreserving the harvestedTIL population; (g) administering a therapeutically effective amount ofthe harvested TIL population to the patient with double-refractorymetastatic melanoma; and (h) measuring the level of IP-10 in the patientafter administering a therapeutically effective amount of the TILs instep (g). In some embodiments, an increase in the level of IP-10 in step(h) is measured. In some embodiments, the increase in the level of IP-10is indicative of treatment response and/or treatment efficacy of the TILadministration. In some embodiments, the increase in the level of IP-10in step (h) is indicative of treatment efficacy. In some embodiments,the level of IP-10 is measured about 1 day to 10 days post administeringthe therapeutically effective amount of the TILs in step (g). In someembodiments, the level of IP-10 is measured 1 day post administering atherapeutically effective amount of the TILs in step (g). In someembodiments, the level of IP-10 is measured about 6 hours to 24 hourspost administering the therapeutically effective amount of the TILs instep (g). In some embodiments, the method further comprises a step ofmeasuring the level of IP-10 in the patient prior to administering atherapeutically effective amount of the TILs in step (g). In someembodiments, the increase is based on an increase in the levels of IP-10in the patient prior to administering a therapeutically effective amountof the TIL in step (g) as compared to the level of IP-10 in the patientprior to administering a therapeutically effective amount of the TILs instep (g). In some embodiments, the method further comprises step (i)predicting the patient will respond to the therapeutically effectiveamount of the TILs administered in step (g) based upon measuring anincrease in the levels of IP-10 in step (h). In some embodiments, themethod further comprises step (i) predicting the patient will notrespond to the therapeutically effective amount of the TILs administeredin step (g) based upon measuring no increase in the levels of IP-10 instep (h). In some embodiments, the method further comprises step (i)predicting the patient will respond to the therapeutically effectiveamount of the TILs administered in step (g) based upon measuring anincrease in the levels of IP-10 in step (h) or predicting the patientwill not respond to the therapeutically effective amount of the TILsadministered in step (g) based upon measuring no increase in the levelsof IP-10 in step (h). In some embodiments, predicting the probabilitythat the patient will or will not respond to the therapeuticallyeffective amount of the TILs administered in step (g) is based upon thepresence or absence of an increase in the level of IP-10 in step (h). Insome embodiments, the increase in the level of IP-10 is an increase ofat least one-fold, two-fold, three-fold, four-fold, five-fold or more.In some embodiments, the level of IP-10 is measured by calculating thedifference between the level of IP-10 before administration of thetherapeutic population of TILs and the level of IP-10 afteradministration of the therapeutic population of TILs. In someembodiments, the level of IP-10 is measured by calculating thedifference between the level of IP-10 at Day −7 before administration ofthe therapeutic population of TILs and the level of IP-10 at Day 1 afteradministration of the therapeutic population of TILs, wherein Day 0 isthe Day of the TIL infusion/administration. In some embodiments,predicting the probability that the patient will or will not respond tothe therapeutically effective amount of the TILs administered in step(g) comprises correlating the level of IP-10 measured in the patientwith a threshold value, wherein if the level of IP-10 measured is abovethe threshold value a further TIL treatment regimen in indicated. Insome embodiments, the threshold value for IP-10 level is about 500 pg/mlto about 3500 pg/mL, wherein an IP-10 level above the threshold value isindicative of treatment efficacy and/or treatment response. In someembodiments, the threshold value for IP-10 level is at least 1000 pg/mL,wherein an IP-10 level above the threshold value is indicative oftreatment efficacy and/or treatment response. In some embodiments, thethreshold value for IP-10 level is at least 1500 pg/mL, wherein an IP-10level above the threshold value is indicative of treatment efficacyand/or treatment response. In some embodiments, the threshold value forIP-10 level is at least 2000 pg/mL, wherein an IP-10 level above thethreshold value is indicative of treatment efficacy and/or treatmentresponse. In some embodiments, the threshold value for IP-10 level is atleast 2500 pg/mL, wherein an IP-10 level above the threshold value isindicative of treatment efficacy and/or treatment response. In someembodiments, the level of IP-10 is about 1000 pg/ml to about 3000 pg/mL,wherein an IP-10 level above the threshold value is indicative oftreatment efficacy and/or treatment response. In some embodiments, thelevel of IP-10 is at least 1000 pg/mL and the IP-10 level is indicativeof treatment efficacy and/or treatment response. In some embodiments,the level of IP-10 is at least 1500 pg/mL and the IP-10 level isindicative of treatment efficacy and/or treatment response. In someembodiments, the level of IP-10 is at least 2000 pg/mL and the IP-10level is indicative of treatment efficacy and/or treatment response. Insome embodiments, the level of IP-10 is at least 2500 pg/mL and theIP-10 level is indicative of treatment efficacy and/or treatmentresponse. In some embodiments, the increase in the level of IP-10 ismeasured by calculating the difference in IP-10 level in plasma sevendays before TIL infusion and one day after TIL infusion, and whereinsaid difference in IP-10 level in plasma is at least 800 pg/mL, at least900 pg/mL, at least 1000 pg/mL, at least 1100 pg/mL, at least 1200pg/mL, at least 1300 pg/mL, at least 1400 pg/mL, at least 1500 pg/mL, atleast 1600 pg/mL, at least 1700 pg/mL, or at least 1800 pg/mL, at least1900 pg/mL, at least 2000 pg/mL, at least 2100 pg/mL, or at least 2200pg/mL. In some embodiments, the difference in IP-10 level in plasma isat least 800 pg/mL. In some embodiments, the difference in IP-10 levelin plasma is at least 900 pg/mL. In some embodiments, the difference inIP-10 level in plasma is at least 1000 pg/mL. In some embodiments, thedifference in IP-10 level in plasma is at least 1100 pg/mL. In someembodiments, the difference in IP-10 level in plasma is at least 1200pg/mL. In some embodiments, the difference in IP-10 level in plasma isat least 1300 pg/mL. In some embodiments, the difference in IP-10 levelin plasma is at least 1400 pg/mL. In some embodiments, the difference inIP-10 level in plasma is at least 1500 pg/mL. In some embodiments, thedifference in IP-10 level in plasma is at least 1600 pg/mL. In someembodiments, the difference in IP-10 level in plasma is at least 1700pg/mL. In some embodiments, the difference in IP-10 level in plasma isor at least 1800 pg/mL. In some embodiments, the difference in IP-10level in plasma is at least 1900 pg/mL. In some embodiments, thedifference in IP-10 level in plasma is at least 2000 pg/mL. In someembodiments, the difference in IP-10 level in plasma is at least 2100pg/mL, or at least 2200 pg/mL. In some embodiments, the difference inIP-10 level in plasma is at least 1600 pg/ml. In some embodiments, thedifference in IP-10 level in plasma is at least 1650 pg/ml. In someembodiments, the difference in IP-10 level in plasma is at least 1656pg/ml. In some embodiments, the increase in the level of IP-10 is atleast one-fold to at least five-fold, as compared to the level of IP-10in the patient before the TIL administration. In some embodiments, theIP-10 is measured before another treatment regimen, such as an anti-PD-1treatment regimen. In some embodiments, the IP-10 is measured afteranother treatment regimen, such as an anti-PD-1 treatment regimen, butbefore administration of TILs. In some embodiments, the level of IP-10is measured before the TILs are harvested for expansion. In someembodiments, the level of IP-10 is measured after the TILs are harvestedfor expansion. In some embodiments, the level IP-10 is measured 6 hoursto 24 hours prior to administration of the TILs to the patient. In someembodiments, the level of IP-10 is measured 1 day, 2 days, 3 days, 4days, 5 days or more before administration of the TILs to the patient.In some embodiments, when there is an increase in the level of IP-10after administration of a therapeutically effective population of tumorinfiltrating lymphocytes, the patient is administered a further dosageof a therapeutically effective population of tumor infiltratinglymphocytes (TILs). In some embodiments, when there is an increase inthe level of IP-10 after administration of a therapeutically effectivepopulation of tumor infiltrating lymphocytes, patient is notadministered a further dosage of a therapeutically effective populationof tumor infiltrating lymphocytes (TILs). In some embodiments, thetherapeutically effective population that results in an increase in thelevel of IP-10 after administration of TILs comprises from about2.3×10¹⁰ to about 13.7×10¹⁰ TILs. In some embodiments, the one, two,three or more therapeutic TIL dosages are administered after determiningthere is an increase in the level of IP-10.

In some embodiments, the invention also provides a method of treatingcancer in a patient in need thereof, the method comprising: (a)obtaining a first population of TILs from a tumor resected from thepatient by processing a tumor sample obtained from the patient intomultiple tumor fragments; (b) adding the tumor fragments into a closedsystem; (c) performing a first expansion by culturing the firstpopulation of TILs in a cell culture medium comprising IL-2, andoptionally OKT-3, to produce a second population of TILs, wherein thefirst expansion is performed in a closed container providing a firstgas-permeable surface area, wherein the first expansion is performed forabout 3-14 days to obtain the second population of TILs, wherein thesecond population of TILs is at least 50-fold greater in number than thefirst population of TILs, and wherein the transition from step (b) tostep (c) occurs without opening the system; (d) performing a secondexpansion by supplementing the cell culture medium of the secondpopulation of TILs with additional IL-2, optionally OKT-3, and antigenpresenting cells (APCs), to produce a third population of TILs, whereinthe second expansion is performed for about 7-14 days to obtain thethird population of TILs, wherein the third population of TILs is atherapeutic population of TILs, wherein the second expansion isperformed in a closed container providing a second gas-permeable surfacearea, and wherein the transition from step (c) to step (d) occurswithout opening the system; (e) harvesting the therapeutic population ofTILs obtained from step (d) to provide a harvested TIL population,wherein the transition from step (d) to step (e) occurs without openingthe system; (f) transferring the harvested TIL population from step (e)to an infusion bag, wherein the transfer from step (e) to (f) occurswithout opening the system, and optionally cryopreserving the harvestedTIL population; (g) administering a therapeutically effective amount ofthe harvested TIL population to the patient with double-refractorymetastatic melanoma; and (h) measuring the level of IP-10 in the patientafter administering a therapeutically effective amount of the TILs instep (g). In some embodiments, an increase in the level of IP-10 in step(h) is measured. In some embodiments, an increase in the level of IP-10in step (h) is indicative of treatment efficacy. In some embodiments,the level of IP-10 is measured about 1 day to 10 days post administeringthe therapeutically effective amount of the TILs in step (g). In someembodiments, the level of IP-10 is measured 1 day post administering atherapeutically effective amount of the TILs in step (g). In someembodiments, the level of IP-10 is measured about 6 hours to 24 hourspost administering the therapeutically effective amount of the TILs instep (g). In some embodiments, the increase in the level of IP-10 is atleast one-fold to at least five-fold, as compared to the level of IP-10in the patient before the TIL administration. In some embodiments, theIP-10 is measured before another treatment regimen, such as an anti-PD-1treatment regimen. In some embodiments, the IP-10 is measured afteranother treatment regimen, such as an anti-PD-1 treatment regimen, butbefore administration of TILs. In some embodiments, the level of IP-10is measured before the TILs are harvested for expansion. In someembodiments, the level of IP-10 is measured after the TILs are harvestedfor expansion. In some embodiments, the level IP-10 is measured 6 hoursto 24 hours prior to administration of the TILs to the patient. In someembodiments, the level of IP-10 is measured 1 day, 2 days, 3 days, 4days, 5 days or more before administration of the TILs to the patient.In some embodiments, the method further comprises a step of measuringthe level of IP-10 in the patient prior to administering atherapeutically effective amount of the TILs in step (g). In someembodiments, the increase is based on an increase in the levels of IP-10in the patient after administering a therapeutically effective amount ofthe TILs in step (g) as compared to the level of IP-10 in the patientprior to administering a therapeutically effective amount of the TILs instep (g). In some embodiments, the method further comprises step (i)predicting the patient will respond to the therapeutically effectiveamount of the TILs administered in step (g) based upon measuring anincrease in the levels of IP-10 in step (h). In some embodiments, themethod further comprises step (i) predicting the patient will notrespond to the therapeutically effective amount of the TILs administeredin step (g) based upon measuring no increase in the levels of IP-10 instep (h). In some embodiments, the level of IP-10 is measured bycalculating the difference between the level of IP-10 beforeadministration of the therapeutic population of TILs and the level ofIP-10 after administration of the therapeutic population of TILs. Insome embodiments, the level of IP-10 is measured by calculating thedifference between the level of IP-10 at Day −7 before administration ofthe therapeutic population of TILs and the level of IP-10 at Day 1 afteradministration of the therapeutic population of TILs, wherein Day 0 isthe Day of the TIL infusion/administration. In some embodiments, thethreshold value for IP-10 level is about 500 pg/ml to about 3500 pg/mL,wherein an IP-10 level above the threshold value is indicative oftreatment efficacy and/or treatment response. In some embodiments, thethreshold value for IP-10 level is at least 1000 pg/mL, wherein an IP-10level above the threshold value is indicative of treatment efficacyand/or treatment response. In some embodiments, the threshold value forIP-10 level is at least 1500 pg/mL, wherein an IP-10 level above thethreshold value is indicative of treatment efficacy and/or treatmentresponse. In some embodiments, the threshold value for IP-10 level is atleast 2000 pg/mL, wherein an IP-10 level above the threshold value isindicative of treatment efficacy and/or treatment response. In someembodiments, the threshold value for IP-10 level is at least 2500 pg/mL,wherein an IP-10 level above the threshold value is indicative oftreatment efficacy and/or treatment response. In some embodiments, thelevel of IP-10 is about 1000 pg/ml to about 3000 pg/mL, wherein an IP-10level above the threshold value is indicative of treatment efficacyand/or treatment response. In some embodiments, the level of IP-10 is atleast 1000 pg/mL and the IP-10 level is indicative of treatment efficacyand/or treatment response. In some embodiments, the level of IP-10 is atleast 1500 pg/mL and the IP-10 level is indicative of treatment efficacyand/or treatment response. In some embodiments, the level of IP-10 is atleast 2000 pg/mL and the IP-10 level is indicative of treatment efficacyand/or treatment response. In some embodiments, the level of IP-10 is atleast 2500 pg/mL and the IP-10 level is indicative of treatment efficacyand/or treatment response. In some embodiments, the increase in thelevel of IP-10 is measured by calculating the difference in IP-10 levelin plasma seven days before TIL infusion and one day after TIL infusion,and wherein said difference in IP-10 level in plasma is at least 800pg/mL, at least 900 pg/mL, at least 1000 pg/mL, at least 1100 pg/mL, atleast 1200 pg/mL, at least 1300 pg/mL, at least 1400 pg/mL, at least1500 pg/mL, at least 1600 pg/mL, at least 1700 pg/mL, or at least 1800pg/mL, at least 1900 pg/mL, at least 2000 pg/mL, at least 2100 pg/mL, orat least 2200 pg/mL. In some embodiments, the difference in IP-10 levelin plasma is at least 800 pg/mL. In some embodiments, the difference inIP-10 level in plasma is at least 900 pg/mL. In some embodiments, thedifference in IP-10 level in plasma is at least 1000 pg/mL. In someembodiments, the difference in IP-10 level in plasma is at least 1100pg/mL. In some embodiments, the difference in IP-10 level in plasma isat least 1200 pg/mL. In some embodiments, the difference in IP-10 levelin plasma is at least 1300 pg/mL. In some embodiments, the difference inIP-10 level in plasma is at least 1400 pg/mL. In some embodiments, thedifference in IP-10 level in plasma is at least 1500 pg/mL. In someembodiments, the difference in IP-10 level in plasma is at least 1600pg/mL. In some embodiments, the difference in IP-10 level in plasma isat least 1700 pg/mL. In some embodiments, the difference in IP-10 levelin plasma is or at least 1800 pg/mL. In some embodiments, the differencein IP-10 level in plasma is at least 1900 pg/mL. In some embodiments,the difference in IP-10 level in plasma is at least 2000 pg/mL. In someembodiments, the difference in IP-10 level in plasma is at least 2100pg/mL, or at least 2200 pg/mL. In some embodiments, the difference inIP-10 level in plasma is at least 1600 pg/ml. In some embodiments, thedifference in IP-10 level in plasma is at least 1650 pg/ml. In someembodiments, the difference in IP-10 level in plasma is at least 1656pg/ml.

In some embodiments, the method further comprises step (i) predictingthe patient will respond to the therapeutically effective amount of theTILs administered in step (g) based upon measuring an increase in thelevels of IP-10 in step (h) or predicting the patient will not respondto the therapeutically effective amount of the TILs administered in step(g) based upon measuring no increase in the levels of IP-10 in step (h).In some embodiments, predicting the probability that the patient will orwill not respond to the therapeutically effective amount of the TILsadministered in step (g) is based upon the presence or absence of anincrease in the level of IP-10 in step (h). In some embodiments, theincrease in the level of IP-10 is an increase of at least one-fold,two-fold, three-fold, four-fold, five-fold or more. In some embodiments,the increase in the level of IP-10 is at least one-fold to at leastfive-fold, as compared to the level of IP-10 in the patient before theTIL administration. In some embodiments, the IP-10 is measured beforeanother treatment regimen, such as an anti-PD-1 treatment regimen. Insome embodiments, the IP-10 is measured after another treatment regimen,such as an anti-PD-1 treatment regimen, but before administration ofTILs. In some embodiments, the level of IP-10 is measured before theTILs are harvested for expansion. In some embodiments, the level ofIP-10 is measured after the TILs are harvested for expansion. In someembodiments, the level IP-10 is measured 6 hours to 24 hours prior toadministration of the TILs to the patient. In some embodiments, thelevel of IP-10 is measured 1 day, 2 days, 3 days, 4 days, 5 days or morebefore administration of the TILs to the patient. In some embodiments,the level of IP-10 is measured by calculating the difference between thelevel of IP-10 before administration of the therapeutic population ofTILs and the level of IP-10 after administration of the therapeuticpopulation of TILs. In some embodiments, the level of IP-10 is measuredby calculating the difference between the level of IP-10 at Day −7before administration of the therapeutic population of TILs and thelevel of IP-10 at Day 1 after administration of the therapeuticpopulation of TILs, wherein Day 0 is the Day of the TILinfusion/administration. In some embodiments, predicting the probabilitythat the patient will or will not respond to the therapeuticallyeffective amount of the TILs administered in step (g) comprisescorrelating the level of IP-10 measured in the patient with a thresholdvalue, wherein if the level of IP-10 measured is above the thresholdvalue a further TIL treatment regimen in indicated. In some embodiments,the threshold value for IP-10 level is about 500 pg/ml to about 3500pg/mL, wherein an IP-10 level above the threshold value is indicative oftreatment efficacy and/or treatment response. In some embodiments, thethreshold value for IP-10 level is at least 1000 pg/mL, wherein an IP-10level above the threshold value is indicative of treatment efficacyand/or treatment response. In some embodiments, the threshold value forIP-10 level is at least 1500 pg/mL, wherein an IP-10 level above thethreshold value is indicative of treatment efficacy and/or treatmentresponse. In some embodiments, the threshold value for IP-10 level is atleast 2000 pg/mL, wherein an IP-10 level above the threshold value isindicative of treatment efficacy and/or treatment response. In someembodiments, the threshold value for IP-10 level is at least 2500 pg/mL,wherein an IP-10 level above the threshold value is indicative oftreatment efficacy and/or treatment response. In some embodiments, thelevel of IP-10 is about 1000 pg/ml to about 3000 pg/mL, wherein an IP-10level above the threshold value is indicative of treatment efficacyand/or treatment response. In some embodiments, the level of IP-10 is atleast 1000 pg/mL and the IP-10 level is indicative of treatment efficacyand/or treatment response. In some embodiments, the level of IP-10 is atleast 1500 pg/mL and the IP-10 level is indicative of treatment efficacyand/or treatment response. In some embodiments, the level of IP-10 is atleast 2000 pg/mL and the IP-10 level is indicative of treatment efficacyand/or treatment response. In some embodiments, the level of IP-10 is atleast 2500 pg/mL and the IP-10 level is indicative of treatment efficacyand/or treatment response. In some embodiments, the level of IP-10 is atleast 2500 pg/mL and the IP-10 level is indicative of treatment efficacyand/or treatment response. In some embodiments, the increase in thelevel of IP-10 is measured by calculating the difference in IP-10 levelin plasma seven days before TIL infusion and one day after TIL infusion,and wherein said difference in IP-10 level in plasma is at least 800pg/mL, at least 900 pg/mL, at least 1000 pg/mL, at least 1100 pg/mL, atleast 1200 pg/mL, at least 1300 pg/mL, at least 1400 pg/mL, at least1500 pg/mL, at least 1600 pg/mL, at least 1700 pg/mL, or at least 1800pg/mL, at least 1900 pg/mL, at least 2000 pg/mL, at least 2100 pg/mL, orat least 2200 pg/mL. In some embodiments, the difference in IP-10 levelin plasma is at least 800 pg/mL. In some embodiments, the difference inIP-10 level in plasma is at least 900 pg/mL. In some embodiments, thedifference in IP-10 level in plasma is at least 1000 pg/mL. In someembodiments, the difference in IP-10 level in plasma is at least 1100pg/mL. In some embodiments, the difference in IP-10 level in plasma isat least 1200 pg/mL. In some embodiments, the difference in IP-10 levelin plasma is at least 1300 pg/mL. In some embodiments, the difference inIP-10 level in plasma is at least 1400 pg/mL. In some embodiments, thedifference in IP-10 level in plasma is at least 1500 pg/mL. In someembodiments, the difference in IP-10 level in plasma is at least 1600pg/mL. In some embodiments, the difference in IP-10 level in plasma isat least 1700 pg/mL. In some embodiments, the difference in IP-10 levelin plasma is or at least 1800 pg/mL. In some embodiments, the differencein IP-10 level in plasma is at least 1900 pg/mL. In some embodiments,the difference in IP-10 level in plasma is at least 2000 pg/mL. In someembodiments, the difference in IP-10 level in plasma is at least 2100pg/mL, or at least 2200 pg/mL. In some embodiments, the difference inIP-10 level in plasma is at least 1600 pg/ml. In some embodiments, thedifference in IP-10 level in plasma is at least 1650 pg/ml. In someembodiments, the difference in IP-10 level in plasma is at least 1656pg/ml. In some embodiments, when there is an increase in the level ofIP-10 after administration of a therapeutically effective population oftumor infiltrating lymphocytes, the patient is administered a furtherdosage of a therapeutically effective population of tumor infiltratinglymphocytes (TILs). In some embodiments, when there is an increase inthe level of IP-10 after administration of a therapeutically effectivepopulation of tumor infiltrating lymphocytes, patient is notadministered a further dosage of a therapeutically effective populationof tumor infiltrating lymphocytes (TILs). In some embodiments, thetherapeutically effective population that results in an increase in thelevel of IP-10 after administration of TILs comprises from about2.3×10¹⁰ to about 13.7×10¹⁰ TILs. In some embodiments, the one, two,three or more therapeutic TIL dosages are administered after determiningthere is an increase in the level of IP-10.

In some embodiments, the invention provides methods of predicting atreatment response and/or predicting treatment efficacy foradministration of a therapeutically effective amount of tumorinfiltrating lymphocytes (TILs) to a patient, the method comprising: a)obtaining a biological sample from a patient with cancer, includingdouble-refractory metastatic melanoma; b) measuring the level of IP-10in the biological sample from a); c) administering a therapeuticallyeffective amount of TILs; d) obtaining a biological sample from thepatient after the administration of the therapeutically effective amountof TILs in step c); e) measuring the level of IP-10 in the biologicalsample from d); f) predicting a treatment response to and/or predictingtreatment efficacy of the administration of the therapeuticallyeffective amount of the TILs based upon the level of IP-10 measuredafter administration as compared to the level of IP-10 measured prior toadministration. In some embodiments, the increase in the level of IP-10measured in step (e) is determined as compared to the level of IP-10measured step (b) is observed. In some embodiments, the increase in thelevel of IP-10 in step (e) is determined as compared to the level ofIP-10 measured step (b) is indicative of treatment efficacy. In someembodiments, the increase in the level of IP-10 is measured in step (e)about 1 day to 10 days post administering a therapeutically effectiveamount of the TILs in step (c). In some embodiments, the increase in thelevel of IP-10 is measured in step (e) about 1 day post administering atherapeutically effective amount of the TILs in step (c). In someembodiments, the increase in the level of IP-10 is measured in step (e)about 6 hours to 24 hours post administering a therapeutically effectiveamount of the TILs in step (g). In some embodiments, predicting that thepatient will or will not respond to the therapeutically effective amountof the TILs administered in step (c) is based upon an increase in thelevel of IP-10 measured in step (f). In some embodiments, detecting anincrease in the level of IP-10 measured in step (e) as compared to thelevel of IP-10 measured step (b) indicates that the patient will respondto the therapeutically effective amount of the TILs administered in step(d). In some embodiments, detecting no increase in the level of IP-10measured in step (e) as compared to the level of IP-10 measured step (b)indicates that the patient will not respond to the therapeuticallyeffective amount of the TILs administered in step (d). In someembodiments, the level of IP-10 is measured by calculating thedifference between the level of IP-10 before administration of thetherapeutic population of TILs and the level of IP-10 afteradministration of the therapeutic population of TILs. In someembodiments, the level of IP-10 is measured by calculating thedifference between the level of IP-10 at Day −7 before administration ofthe therapeutic population of TILs and the level of IP-10 at Day 1 afteradministration of the therapeutic population of TILs, wherein Day 0 isthe Day of the TIL infusion/administration. In some embodiments, thelevel of IP-10 is increased one-fold, two-fold, three-fold, four-fold,five-fold or more. In some embodiments, the increase in the level ofIP-10 is at least one-fold to at least five-fold, as compared to thelevel of IP-10 in the patient before the TIL administration. In someembodiments, the IP-10 is measured before another treatment regimen,such as an anti-PD-1 treatment regimen. In some embodiments, the IP-10is measured after another treatment regimen, such as an anti-PD-1treatment regimen, but before administration of TILs. In someembodiments, the level of IP-10 is measured before the TILs areharvested for expansion. In some embodiments, the level of IP-10 ismeasured after the TILs are harvested for expansion. In someembodiments, the level IP-10 is measured 6 hours to 24 hours prior toadministration of the TILs to the patient. In some embodiments, thelevel of IP-10 is measured 1 day, 2 days, 3 days, 4 days, 5 days or morebefore administration of the TILs to the patient. In some embodiments,predicting that the patient will or will not respond to thetherapeutically effective amount of the TILs administered in step (d)further comprises correlating the level of IP-10 measured in the patientwith a threshold value. In some embodiments, the threshold value forIP-10 level is about 500 pg/ml to about 3500 pg/mL, wherein an IP-10level above the threshold value is indicative of treatment efficacyand/or treatment response. In some embodiments, the threshold value forIP-10 level is at least 1000 pg/mL, wherein an IP-10 level above thethreshold value is indicative of treatment efficacy and/or treatmentresponse. In some embodiments, the threshold value for IP-10 level is atleast 1500 pg/mL, wherein an IP-10 level above the threshold value isindicative of treatment efficacy and/or treatment response. In someembodiments, the threshold value for IP-10 level is at least 2000 pg/mL,wherein an IP-10 level above the threshold value is indicative oftreatment efficacy and/or treatment response. In some embodiments, thethreshold value for IP-10 level is at least 2500 pg/mL, wherein an IP-10level above the threshold value is indicative of treatment efficacyand/or treatment response. In some embodiments, the level of IP-10 isabout 1000 pg/ml to about 3000 pg/mL, wherein an IP-10 level above thethreshold value is indicative of treatment efficacy and/or treatmentresponse. In some embodiments, the level of IP-10 is at least 1000 pg/mLand the IP-10 level is indicative of treatment efficacy and/or treatmentresponse. In some embodiments, the level of IP-10 is at least 1500 pg/mLand the IP-10 level is indicative of treatment efficacy and/or treatmentresponse. In some embodiments, the level of IP-10 is at least 2000 pg/mLand the IP-10 level is indicative of treatment efficacy and/or treatmentresponse. In some embodiments, the level of IP-10 is at least 2500 pg/mLand the IP-10 level is indicative of treatment efficacy and/or treatmentresponse. In some embodiments, the level of IP-10 is at least 2500 pg/mLand the IP-10 level is indicative of treatment efficacy and/or treatmentresponse. In some embodiments, the increase in the level of IP-10 ismeasured by calculating the difference in IP-10 level in plasma sevendays before TIL infusion and one day after TIL infusion, and whereinsaid difference in IP-10 level in plasma is at least 800 pg/mL, at least900 pg/mL, at least 1000 pg/mL, at least 1100 pg/mL, at least 1200pg/mL, at least 1300 pg/mL, at least 1400 pg/mL, at least 1500 pg/mL, atleast 1600 pg/mL, at least 1700 pg/mL, or at least 1800 pg/mL, at least1900 pg/mL, at least 2000 pg/mL, at least 2100 pg/mL, or at least 2200pg/mL. In some embodiments, the difference in IP-10 level in plasma isat least 800 pg/mL. In some embodiments, the difference in IP-10 levelin plasma is at least 900 pg/mL. In some embodiments, the difference inIP-10 level in plasma is at least 1000 pg/mL. In some embodiments, thedifference in IP-10 level in plasma is at least 1100 pg/mL. In someembodiments, the difference in IP-10 level in plasma is at least 1200pg/mL. In some embodiments, the difference in IP-10 level in plasma isat least 1300 pg/mL. In some embodiments, the difference in IP-10 levelin plasma is at least 1400 pg/mL. In some embodiments, the difference inIP-10 level in plasma is at least 1500 pg/mL. In some embodiments, thedifference in IP-10 level in plasma is at least 1600 pg/mL. In someembodiments, the difference in IP-10 level in plasma is at least 1700pg/mL. In some embodiments, the difference in IP-10 level in plasma isor at least 1800 pg/mL. In some embodiments, the difference in IP-10level in plasma is at least 1900 pg/mL. In some embodiments, thedifference in IP-10 level in plasma is at least 2000 pg/mL. In someembodiments, the difference in IP-10 level in plasma is at least 2100pg/mL, or at least 2200 pg/mL. In some embodiments, the difference inIP-10 level in plasma is at least 1600 pg/ml. In some embodiments, thedifference in IP-10 level in plasma is at least 1650 pg/ml. In someembodiments, the difference in IP-10 level in plasma is at least 1656pg/ml. In some embodiments, when there is an increase in the level ofIP-10 after administration of a therapeutically effective population oftumor infiltrating lymphocytes, the patient is administered one or morefurther dosages of a therapeutically effective population of tumorinfiltrating lymphocytes (TILs). In some embodiments, when there is anincrease in the level of IP-10 after administration of a therapeuticallyeffective population of tumor infiltrating lymphocytes, patient is notadministered a further dosage of a therapeutically effective populationof tumor infiltrating lymphocytes (TILs). In some embodiments, thetherapeutically effective population that results in an increase in thelevel of IP-10 after administration of TILs comprises from about2.3×10¹⁰ to about 13.7×10¹⁰ TILs. In some embodiments, the one, two,three or more therapeutic TIL dosages are administered after determiningthere is an increase in the level of IP-10.

According to the methods described herein, IP-10 production can bemeasured by determining the levels of the IP-10 in the blood of asubject treated with TILs prepared by the methods of the presentinvention, including those as described for example in FIG. 18. In someembodiments, the level of IP-10 is the level of IP-10 protein in asample. In some embodiments, IP-10 is measured by a commercial Bio-Radbead-based Bio-Plex immunoassay, which measures multiple cytokines andchemokines, and which includes an antibody specific for IP-10. In someembodiments, IP-10 is measured by taking blood samples from the patientand is measured in the plasma fraction obtained from the blood (i.e.,after all blood cells are removed) and is reported in units of picogramsper milliliter of plasma. In some embodiments, the level of IP-10 ismeasured by calculating the difference between the level of IP-10 beforeadministration of the therapeutic population of TILs and the level ofIP-10 after administration of the therapeutic population of TILs. Insome embodiments, the level of IP-10 is measured by calculating thedifference between the level of IP-10 at Day −7 before administration ofthe therapeutic population of TILs and the level of IP-10 at Day 1 afteradministration of the therapeutic population of TILs, wherein Day 0 isthe Day of the TIL infusion/administration. In some embodiments, higherIP-10 is indicative of treatment efficacy and/or increased clinicalefficacy. In some embodiments, higher IP-10 in the blood of subjectstreated with TILs is indicative of active TILs. IP-10 production can bemeasured by determining the levels of the IP-10 in the blood of asubject treated with TILs prepared by the methods of the presentinvention, including those as described for example in FIG. 18. In someembodiments, higher IP-10 is indicative of treatment efficacy in apatient treated with the TILs produced by the methods of the presentinvention. In some embodiments, higher IP-10 correlates to an increaseof one-fold, two-fold, three-fold, four-fold, or five-fold or more ascompared to an untreated patient and/or as compared to a patient treatedwith TILs prepared using other methods than those provide hereinincluding for example, methods other than those embodied in FIG. 18. Insome embodiments, higher IP-10 correlates to an increase of one-fold ascompared to an untreated patient and/or as compared to a patient treatedwith TILs prepared using other methods than those provide hereinincluding for example, methods other than those embodied in FIG. 18. Insome embodiments, higher IP-10 correlates to an increase of two-fold ascompared to an untreated patient and/or as compared to a patient treatedwith TILs prepared using other methods than those provide hereinincluding for example, methods other than those embodied in FIG. 18. Insome embodiments, higher IP-10 correlates to an increase of three-foldas compared to an untreated patient and/or as compared to a patienttreated with TILs prepared using other methods than those provide hereinincluding for example, methods other than those embodied in FIG. 18. Insome embodiments, higher IP-10 correlates to an increase of four-fold ascompared to an untreated patient and/or as compared to a patient treatedwith TILs prepared using other methods than those provide hereinincluding for example, methods other than those embodied in FIG. 18. Insome embodiments, higher IP-10 correlates to an increase of five-fold ascompared to an untreated patient and/or as compared to a patient treatedwith TILs prepared using other methods than those provide hereinincluding for example, methods other than those embodied in FIG. 18. Insome embodiments, IP-10 is measured in blood of a subject treated withTILs prepared by the methods of the present invention, including thoseas described for example in FIG. 18. In some embodiments, IP-10 ismeasured in TILs serum of a subject treated with TILs prepared by themethods of the present invention, including those as described forexample in FIG. 18.

Certain Exemplary Embodiments

In an embodiment, the invention provides a method of treating a cancerwith a population of tumor infiltrating lymphocytes (TILs) comprisingthe steps of:

-   -   (a) resecting a tumor from a patient, the tumor comprising a        first population of TILs;    -   (b) fragmenting the tumor into tumor fragments;    -   (c) contacting the tumor fragments with a first cell culture        medium;    -   (d) performing an initial expansion of the first population of        TILs in the first cell culture medium to obtain a second        population of TILs, wherein the second population of TILs is at        least 5-fold greater in number than the first population of        TILs, wherein the first cell culture medium comprises IL-2;    -   (e) performing a rapid expansion of the second population of        TILs in a second cell culture medium to obtain a third        population of TILs, wherein the third population of TILs is at        least 50-fold greater in number than the second population of        TILs after 7 days from the start of the rapid expansion; wherein        the second cell culture medium comprises IL-2, OKT-3 (anti-CD3        antibody), and irradiated allogeneic peripheral blood        mononuclear cells (PBMCs); and wherein the rapid expansion is        performed over a period of 14 days or less;    -   (f) harvesting the third population of TILs; and    -   (g) administering a therapeutically effective portion of the        third population of TILs to a patient with the cancer;    -   wherein the cancer is double-refractory metastatic melanoma.

In an embodiment, the invention provides a method of treating a cancerwith a population of tumor infiltrating lymphocytes (TILs) comprisingthe steps of:

-   -   (a) resecting a tumor from a patient, the tumor comprising a        first population of TILs;    -   (b) fragmenting the tumor into tumor fragments;    -   (c) contacting the tumor fragments with a first cell culture        medium;    -   (d) performing an initial expansion of the first population of        TILs in the first cell culture medium to obtain a second        population of TILs, wherein the second population of TILs is at        least 5-fold greater in number than the first population of        TILs, wherein the first cell culture medium comprises IL-2;    -   (e) performing a rapid expansion of the second population of        TILs in a second cell culture medium to obtain a third        population of TILs, wherein the third population of TILs is at        least 50-fold greater in number than the second population of        TILs after 7 days from the start of the rapid expansion; wherein        the second cell culture medium comprises IL-2, OKT-3 (anti-CD3        antibody), and irradiated allogeneic peripheral blood        mononuclear cells (PBMCs); and wherein the rapid expansion is        performed over a period of 14 days or less;    -   (f) harvesting the third population of TILs; and    -   (g) administering a therapeutically effective portion of the        third population of TILs to a patient with the cancer;    -   wherein the cancer is double-refractory metastatic melanoma,        wherein the double-refractory metastatic melanoma is a cutaneous        double-refractory metastatic melanoma.

In an embodiment, the invention provides a method of treating a cancerwith a population of tumor infiltrating lymphocytes (TILs) comprisingthe steps of:

-   -   (a) resecting a tumor from a patient, the tumor comprising a        first population of TILs;    -   (b) fragmenting the tumor into tumor fragments;    -   (c) contacting the tumor fragments with a first cell culture        medium;    -   (d) performing an initial expansion of the first population of        TILs in the first cell culture medium to obtain a second        population of TILs, wherein the second population of TILs is at        least 5-fold greater in number than the first population of        TILs, wherein the first cell culture medium comprises IL-2;    -   (e) performing a rapid expansion of the second population of        TILs in a second cell culture medium to obtain a third        population of TILs, wherein the third population of TILs is at        least 50-fold greater in number than the second population of        TILs after 7 days from the start of the rapid expansion; wherein        the second cell culture medium comprises IL-2, OKT-3 (anti-CD3        antibody), and irradiated allogeneic peripheral blood        mononuclear cells (PBMCs); and wherein the rapid expansion is        performed over a period of 14 days or less;    -   (f) harvesting the third population of TILs; and    -   (g) administering a therapeutically effective portion of the        third population of TILs to a patient with the cancer;    -   wherein the cancer is double-refractory metastatic melanoma,        wherein the double-refractory metastatic melanoma is refractory        to at least two prior systemic treatment courses, not including        neo-adjuvant or adjuvant therapies.

In an embodiment, the invention provides a method of treating a cancerwith a population of tumor infiltrating lymphocytes (TILs) comprisingthe steps of:

-   -   (a) resecting a tumor from a patient, the tumor comprising a        first population of TILs;    -   (b) fragmenting the tumor into tumor fragments;    -   (c) contacting the tumor fragments with a first cell culture        medium;    -   (d) performing an initial expansion of the first population of        TILs in the first cell culture medium to obtain a second        population of TILs, wherein the second population of TILs is at        least 5-fold greater in number than the first population of        TILs, wherein the first cell culture medium comprises IL-2;    -   (e) performing a rapid expansion of the second population of        TILs in a second cell culture medium to obtain a third        population of TILs, wherein the third population of TILs is at        least 50-fold greater in number than the second population of        TILs after 7 days from the start of the rapid expansion; wherein        the second cell culture medium comprises IL-2, OKT-3 (anti-CD3        antibody), and irradiated allogeneic peripheral blood        mononuclear cells (PBMCs); and wherein the rapid expansion is        performed over a period of 14 days or less;    -   (f) harvesting the third population of TILs; and    -   (g) administering a therapeutically effective portion of the        third population of TILs to a patient with the cancer;    -   wherein the cancer is double-refractory metastatic melanoma,        wherein the double-refractory metastatic melanoma is refractory        to aldesleukin or a biosimilar thereof.

In an embodiment, the invention provides a method of treating a cancerwith a population of tumor infiltrating lymphocytes (TILs) comprisingthe steps of:

-   -   (a) resecting a tumor from a patient, the tumor comprising a        first population of TILs;    -   (b) fragmenting the tumor into tumor fragments;    -   (c) contacting the tumor fragments with a first cell culture        medium;    -   (d) performing an initial expansion of the first population of        TILs in the first cell culture medium to obtain a second        population of TILs, wherein the second population of TILs is at        least 5-fold greater in number than the first population of        TILs, wherein the first cell culture medium comprises IL-2;    -   (e) performing a rapid expansion of the second population of        TILs in a second cell culture medium to obtain a third        population of TILs, wherein the third population of TILs is at        least 50-fold greater in number than the second population of        TILs after 7 days from the start of the rapid expansion; wherein        the second cell culture medium comprises IL-2, OKT-3 (anti-CD3        antibody), and irradiated allogeneic peripheral blood        mononuclear cells (PBMCs); and wherein the rapid expansion is        performed over a period of 14 days or less;    -   (f) harvesting the third population of TILs; and    -   (g) administering a therapeutically effective portion of the        third population of TILs to a patient with the cancer;    -   wherein the cancer is double-refractory metastatic melanoma,        wherein the double-refractory metastatic melanoma is refractory        to pembrolizumab or a biosimilar thereof.

In an embodiment, the invention provides a method of treating a cancerwith a population of tumor infiltrating lymphocytes (TILs) comprisingthe steps of:

-   -   (a) resecting a tumor from a patient, the tumor comprising a        first population of TILs;    -   (b) fragmenting the tumor into tumor fragments;    -   (c) contacting the tumor fragments with a first cell culture        medium;    -   (d) performing an initial expansion of the first population of        TILs in the first cell culture medium to obtain a second        population of TILs, wherein the second population of TILs is at        least 5-fold greater in number than the first population of        TILs, wherein the first cell culture medium comprises IL-2;    -   (e) performing a rapid expansion of the second population of        TILs in a second cell culture medium to obtain a third        population of TILs, wherein the third population of TILs is at        least 50-fold greater in number than the second population of        TILs after 7 days from the start of the rapid expansion; wherein        the second cell culture medium comprises IL-2, OKT-3 (anti-CD3        antibody), and irradiated allogeneic peripheral blood        mononuclear cells (PBMCs); and wherein the rapid expansion is        performed over a period of 14 days or less;    -   (f) harvesting the third population of TILs; and    -   (g) administering a therapeutically effective portion of the        third population of TILs to a patient with the cancer;    -   wherein the cancer is double-refractory metastatic melanoma,        wherein the double-refractory metastatic melanoma is refractory        to nivolumab or a biosimilar thereof.    -   (a) resecting a tumor from a patient, the tumor comprising a        first population of TILs;    -   (b) fragmenting the tumor into tumor fragments;    -   (c) contacting the tumor fragments with a first cell culture        medium;    -   (d) performing an initial expansion of the In an embodiment, the        invention provides a method of treating a cancer with a        population of tumor infiltrating lymphocytes (TILs) comprising        the steps of:    -   first population of TILs in the first cell culture medium to        obtain a second population of TILs, wherein the second        population of TILs is at least 5-fold greater in number than the        first population of TILs, wherein the first cell culture medium        comprises IL-2;    -   (e) performing a rapid expansion of the second population of        TILs in a second cell culture medium to obtain a third        population of TILs, wherein the third population of TILs is at        least 50-fold greater in number than the second population of        TILs after 7 days from the start of the rapid expansion; wherein        the second cell culture medium comprises IL-2, OKT-3 (anti-CD3        antibody), and irradiated allogeneic peripheral blood        mononuclear cells (PBMCs); and wherein the rapid expansion is        performed over a period of 14 days or less;    -   (f) harvesting the third population of TILs; and    -   (g) administering a therapeutically effective portion of the        third population of TILs to a patient with the cancer;    -   wherein the cancer is double-refractory metastatic melanoma,        wherein the double-refractory metastatic melanoma is refractory        to ipilimumab or a biosimilar thereof.

In an embodiment, the invention provides a method of treating a cancerwith a population of tumor infiltrating lymphocytes (TILs) comprisingthe steps of:

-   -   (a) resecting a tumor from a patient, the tumor comprising a        first population of TILs;    -   (b) fragmenting the tumor into tumor fragments;    -   (c) contacting the tumor fragments with a first cell culture        medium;    -   (d) performing an initial expansion of the first population of        TILs in the first cell culture medium to obtain a second        population of TILs, wherein the second population of TILs is at        least 5-fold greater in number than the first population of        TILs, wherein the first cell culture medium comprises IL-2;    -   (e) performing a rapid expansion of the second population of        TILs in a second cell culture medium to obtain a third        population of TILs, wherein the third population of TILs is at        least 50-fold greater in number than the second population of        TILs after 7 days from the start of the rapid expansion; wherein        the second cell culture medium comprises IL-2, OKT-3 (anti-CD3        antibody), and irradiated allogeneic peripheral blood        mononuclear cells (PBMCs); and wherein the rapid expansion is        performed over a period of 14 days or less;    -   (f) harvesting the third population of TILs; and    -   (g) administering a therapeutically effective portion of the        third population of TILs to a patient with the cancer;    -   wherein the cancer is double-refractory metastatic melanoma,        wherein the double-refractory metastatic melanoma is refractory        to ipilimumab or a biosimilar thereof and pembrolizumab or a        biosimilar thereof.

In an embodiment, the invention provides a method of treating a cancerwith a population of tumor infiltrating lymphocytes (TILs) comprisingthe steps of:

-   -   (a) resecting a tumor from a patient, the tumor comprising a        first population of TILs;    -   (b) fragmenting the tumor into tumor fragments;    -   (c) contacting the tumor fragments with a first cell culture        medium;    -   (d) performing an initial expansion of the first population of        TILs in the first cell culture medium to obtain a second        population of TILs, wherein the second population of TILs is at        least 5-fold greater in number than the first population of        TILs, wherein the first cell culture medium comprises IL-2;    -   (e) performing a rapid expansion of the second population of        TILs in a second cell culture medium to obtain a third        population of TILs, wherein the third population of TILs is at        least 50-fold greater in number than the second population of        TILs after 7 days from the start of the rapid expansion; wherein        the second cell culture medium comprises IL-2, OKT-3 (anti-CD3        antibody), and irradiated allogeneic peripheral blood        mononuclear cells (PBMCs); and wherein the rapid expansion is        performed over a period of 14 days or less;    -   (f) harvesting the third population of TILs; and    -   (g) administering a therapeutically effective portion of the        third population of TILs to a patient with the cancer;    -   wherein the cancer is double-refractory metastatic melanoma,        wherein the double-refractory metastatic melanoma is refractory        to ipilimumab or a biosimilar thereof and nivolumab or a        biosimilar thereof.

In an embodiment, the invention provides a method of treating a cancerwith a population of tumor infiltrating lymphocytes (TILs) comprisingthe steps of:

-   -   (a) resecting a tumor from a patient, the tumor comprising a        first population of TILs;    -   (b) fragmenting the tumor into tumor fragments;    -   (c) contacting the tumor fragments with a first cell culture        medium;    -   (d) performing an initial expansion of the first population of        TILs in the first cell culture medium to obtain a second        population of TILs, wherein the second population of TILs is at        least 5-fold greater in number than the first population of        TILs, wherein the first cell culture medium comprises IL-2;    -   (e) performing a rapid expansion of the second population of        TILs in a second cell culture medium to obtain a third        population of TILs, wherein the third population of TILs is at        least 50-fold greater in number than the second population of        TILs after 7 days from the start of the rapid expansion; wherein        the second cell culture medium comprises IL-2, OKT-3 (anti-CD3        antibody), and irradiated allogeneic peripheral blood        mononuclear cells (PBMCs); and wherein the rapid expansion is        performed over a period of 14 days or less;    -   (f) harvesting the third population of TILs; and    -   (g) administering a therapeutically effective portion of the        third population of TILs to a patient with the cancer;    -   wherein the cancer is double-refractory metastatic melanoma,        wherein the initial expansion is performed over a period of 21        days or less.

In an embodiment, the invention provides a method of treating a cancerwith a population of tumor infiltrating lymphocytes (TILs) comprisingthe steps of:

-   -   (a) resecting a tumor from a patient, the tumor comprising a        first population of TILs;    -   (b) fragmenting the tumor into tumor fragments;    -   (c) contacting the tumor fragments with a first cell culture        medium;    -   (d) performing an initial expansion of the first population of        TILs in the first cell culture medium to obtain a second        population of TILs, wherein the second population of TILs is at        least 5-fold greater in number than the first population of        TILs, wherein the first cell culture medium comprises IL-2;    -   (e) performing a rapid expansion of the second population of        TILs in a second cell culture medium to obtain a third        population of TILs, wherein the third population of TILs is at        least 50-fold greater in number than the second population of        TILs after 7 days from the start of the rapid expansion; wherein        the second cell culture medium comprises IL-2, OKT-3 (anti-CD3        antibody), and irradiated allogeneic peripheral blood        mononuclear cells (PBMCs); and wherein the rapid expansion is        performed over a period of 14 days or less;    -   (f) harvesting the third population of TILs; and    -   (g) administering a therapeutically effective portion of the        third population of TILs to a patient with the cancer;    -   wherein the cancer is double-refractory metastatic melanoma,        wherein the initial expansion is performed over a period of 11        days or less.

In an embodiment, the invention provides a method of treating a cancerwith a population of tumor infiltrating lymphocytes (TILs) comprisingthe steps of:

-   -   (a) resecting a tumor from a patient, the tumor comprising a        first population of TILs;    -   (b) fragmenting the tumor into tumor fragments;    -   (c) contacting the tumor fragments with a first cell culture        medium;    -   (d) performing an initial expansion of the first population of        TILs in the first cell culture medium to obtain a second        population of TILs, wherein the second population of TILs is at        least 5-fold greater in number than the first population of        TILs, wherein the first cell culture medium comprises IL-2;    -   (e) performing a rapid expansion of the second population of        TILs in a second cell culture medium to obtain a third        population of TILs, wherein the third population of TILs is at        least 50-fold greater in number than the second population of        TILs after 7 days from the start of the rapid expansion; wherein        the second cell culture medium comprises IL-2, OKT-3 (anti-CD3        antibody), and irradiated allogeneic peripheral blood        mononuclear cells (PBMCs); and wherein the rapid expansion is        performed over a period of 14 days or less;    -   (f) harvesting the third population of TILs; and    -   (g) administering a therapeutically effective portion of the        third population of TILs to a patient with the cancer;    -   wherein the cancer is double-refractory metastatic melanoma,        wherein the IL-2 is present at an initial concentration of        between 1000 IU/mL and 6000 IU/mL in the first cell culture        medium.

In an embodiment, the invention provides a method of treating a cancerwith a population of tumor infiltrating lymphocytes (TILs) comprisingthe steps of:

-   -   (a) resecting a tumor from a patient, the tumor comprising a        first population of TILs;    -   (b) fragmenting the tumor into tumor fragments;    -   (c) contacting the tumor fragments with a first cell culture        medium;    -   (d) performing an initial expansion of the first population of        TILs in the first cell culture medium to obtain a second        population of TILs, wherein the second population of TILs is at        least 5-fold greater in number than the first population of        TILs, wherein the first cell culture medium comprises IL-2;    -   (e) performing a rapid expansion of the second population of        TILs in a second cell culture medium to obtain a third        population of TILs, wherein the third population of TILs is at        least 50-fold greater in number than the second population of        TILs after 7 days from the start of the rapid expansion; wherein        the second cell culture medium comprises IL-2, OKT-3 (anti-CD3        antibody), and irradiated allogeneic peripheral blood        mononuclear cells (PBMCs); and wherein the rapid expansion is        performed over a period of 14 days or less;    -   (f) harvesting the third population of TILs; and    -   (g) administering a therapeutically effective portion of the        third population of TILs to a patient with the cancer;    -   wherein the cancer is double-refractory metastatic melanoma,        wherein the IL-2 is present at an initial concentration of        between 1000 IU/mL and 6000 IU/mL and the OKT-3 antibody is        present at an initial concentration of about 30 ng/mL in the        second cell culture medium.

In an embodiment, the invention provides a method of treating a cancerwith a population of tumor infiltrating lymphocytes (TILs) comprisingthe steps of:

-   -   (a) resecting a tumor from a patient, the tumor comprising a        first population of TILs;    -   (b) fragmenting the tumor into tumor fragments;    -   (c) contacting the tumor fragments with a first cell culture        medium;    -   (d) performing an initial expansion of the first population of        TILs in the first cell culture medium to obtain a second        population of TILs, wherein the second population of TILs is at        least 5-fold greater in number than the first population of        TILs, wherein the first cell culture medium comprises IL-2;    -   (e) performing a rapid expansion of the second population of        TILs in a second cell culture medium to obtain a third        population of TILs, wherein the third population of TILs is at        least 50-fold greater in number than the second population of        TILs after 7 days from the start of the rapid expansion; wherein        the second cell culture medium comprises IL-2, OKT-3 (anti-CD3        antibody), and irradiated allogeneic peripheral blood        mononuclear cells (PBMCs); and wherein the rapid expansion is        performed over a period of 14 days or less;    -   (f) harvesting the third population of TILs; and    -   (g) administering a therapeutically effective portion of the        third population of TILs to a patient with the cancer;    -   wherein the cancer is double-refractory metastatic melanoma,        wherein the initial expansion is performed using a gas permeable        container.

In an embodiment, the invention provides a method of treating a cancerwith a population of tumor infiltrating lymphocytes (TILs) comprisingthe steps of:

-   -   (a) resecting a tumor from a patient, the tumor comprising a        first population of TILs;    -   (b) fragmenting the tumor into tumor fragments;    -   (c) contacting the tumor fragments with a first cell culture        medium;    -   (d) performing an initial expansion of the first population of        TILs in the first cell culture medium to obtain a second        population of TILs, wherein the second population of TILs is at        least 5-fold greater in number than the first population of        TILs, wherein the first cell culture medium comprises IL-2;    -   (e) performing a rapid expansion of the second population of        TILs in a second cell culture medium to obtain a third        population of TILs, wherein the third population of TILs is at        least 50-fold greater in number than the second population of        TILs after 7 days from the start of the rapid expansion; wherein        the second cell culture medium comprises IL-2, OKT-3 (anti-CD3        antibody), and irradiated allogeneic peripheral blood        mononuclear cells (PBMCs); and wherein the rapid expansion is        performed over a period of 14 days or less;    -   (f) harvesting the third population of TILs; and    -   (g) administering a therapeutically effective portion of the        third population of TILs to a patient with the cancer;    -   wherein the cancer is double-refractory metastatic melanoma,        wherein the rapid expansion is performed using a gas permeable        container.

In an embodiment, the invention provides a method of treating a cancerwith a population of tumor infiltrating lymphocytes (TILs) comprisingthe steps of:

-   -   (a) resecting a tumor from a patient, the tumor comprising a        first population of TILs;    -   (b) fragmenting the tumor into tumor fragments;    -   (c) contacting the tumor fragments with a first cell culture        medium;    -   (d) performing an initial expansion of the first population of        TILs in the first cell culture medium to obtain a second        population of TILs, wherein the second population of TILs is at        least 5-fold greater in number than the first population of        TILs, wherein the first cell culture medium comprises IL-2;    -   (e) performing a rapid expansion of the second population of        TILs in a second cell culture medium to obtain a third        population of TILs, wherein the third population of TILs is at        least 50-fold greater in number than the second population of        TILs after 7 days from the start of the rapid expansion; wherein        the second cell culture medium comprises IL-2, OKT-3 (anti-CD3        antibody), and irradiated allogeneic peripheral blood        mononuclear cells (PBMCs); and wherein the rapid expansion is        performed over a period of 14 days or less;    -   (f) harvesting the third population of TILs; and    -   (g) administering a therapeutically effective portion of the        third population of TILs to a patient with the cancer;    -   wherein the cancer is double-refractory metastatic melanoma,        wherein the first cell culture medium further comprises a        cytokine selected from the group consisting of IL-4, IL-7,        IL-15, IL-21, and combinations thereof.

In an embodiment, the invention provides a method of treating a cancerwith a population of tumor infiltrating lymphocytes (TILs) comprisingthe steps of:

-   -   (a) resecting a tumor from a patient, the tumor comprising a        first population of TILs;    -   (b) fragmenting the tumor into tumor fragments;    -   (c) contacting the tumor fragments with a first cell culture        medium;    -   (d) performing an initial expansion of the first population of        TILs in the first cell culture medium to obtain a second        population of TILs, wherein the second population of TILs is at        least 5-fold greater in number than the first population of        TILs, wherein the first cell culture medium comprises IL-2;    -   (e) performing a rapid expansion of the second population of        TILs in a second cell culture medium to obtain a third        population of TILs, wherein the third population of TILs is at        least 50-fold greater in number than the second population of        TILs after 7 days from the start of the rapid expansion; wherein        the second cell culture medium comprises IL-2, OKT-3 (anti-CD3        antibody), and irradiated allogeneic peripheral blood        mononuclear cells (PBMCs); and wherein the rapid expansion is        performed over a period of 14 days or less;    -   (f) harvesting the third population of TILs; and    -   (g) administering a therapeutically effective portion of the        third population of TILs to a patient with the cancer;    -   wherein the cancer is double-refractory metastatic melanoma,        wherein the second cell culture medium further comprises a        cytokine selected from the group consisting of IL-4, IL-7,        IL-15, IL-21, and combinations thereof.

In an embodiment, the invention provides a method of treating a cancerwith a population of tumor infiltrating lymphocytes (TILs) comprisingthe steps of:

-   -   (a) resecting a tumor from a patient, the tumor comprising a        first population of TILs;    -   (b) fragmenting the tumor into tumor fragments;    -   (c) contacting the tumor fragments with a first cell culture        medium;    -   (d) performing an initial expansion of the first population of        TILs in the first cell culture medium to obtain a second        population of TILs, wherein the second population of TILs is at        least 5-fold greater in number than the first population of        TILs, wherein the first cell culture medium comprises IL-2;    -   (e) performing a rapid expansion of the second population of        TILs in a second cell culture medium to obtain a third        population of TILs, wherein the third population of TILs is at        least 50-fold greater in number than the second population of        TILs after 7 days from the start of the rapid expansion; wherein        the second cell culture medium comprises IL-2, OKT-3 (anti-CD3        antibody), and irradiated allogeneic peripheral blood        mononuclear cells (PBMCs); and wherein the rapid expansion is        performed over a period of 14 days or less;    -   (f) harvesting the third population of TILs; and    -   (g) administering a therapeutically effective portion of the        third population of TILs to a patient with the cancer;    -   wherein the cancer is double-refractory metastatic melanoma,        further comprising the step of treating the patient with a        non-myeloablative lymphodepletion regimen prior to administering        the third population of TILs to the patient.

In an embodiment, the invention provides a method of treating a cancerwith a population of tumor infiltrating lymphocytes (TILs) comprisingthe steps of:

-   -   (a) resecting a tumor from a patient, the tumor comprising a        first population of TILs;    -   (b) fragmenting the tumor into tumor fragments;    -   (c) contacting the tumor fragments with a first cell culture        medium;    -   (d) performing an initial expansion of the first population of        TILs in the first cell culture medium to obtain a second        population of TILs, wherein the second population of TILs is at        least 5-fold greater in number than the first population of        TILs, wherein the first cell culture medium comprises IL-2;    -   (e) performing a rapid expansion of the second population of        TILs in a second cell culture medium to obtain a third        population of TILs, wherein the third population of TILs is at        least 50-fold greater in number than the second population of        TILs after 7 days from the start of the rapid expansion; wherein        the second cell culture medium comprises IL-2, OKT-3 (anti-CD3        antibody), and irradiated allogeneic peripheral blood        mononuclear cells (PBMCs); and wherein the rapid expansion is        performed over a period of 14 days or less;    -   (f) harvesting the third population of TILs; and    -   (g) administering a therapeutically effective portion of the        third population of TILs to a patient with the cancer;    -   wherein the cancer is double-refractory metastatic melanoma,        wherein the non-myeloablative lymphodepletion regimen comprises        the steps of administration of cyclophosphamide at a dose of 60        mg/m²/day for two days followed by administration of fludarabine        at a dose of 25 mg/m²/day for five days.

In an embodiment, the invention provides a method of treating a cancerwith a population of tumor infiltrating lymphocytes (TILs) comprisingthe steps of:

-   -   (a) resecting a tumor from a patient, the tumor comprising a        first population of TILs;    -   (b) fragmenting the tumor into tumor fragments;    -   (c) contacting the tumor fragments with a first cell culture        medium;    -   (d) performing an initial expansion of the first population of        TILs in the first cell culture medium to obtain a second        population of TILs, wherein the second population of TILs is at        least 5-fold greater in number than the first population of        TILs, wherein the first cell culture medium comprises IL-2;    -   (e) performing a rapid expansion of the second population of        TILs in a second cell culture medium to obtain a third        population of TILs, wherein the third population of TILs is at        least 50-fold greater in number than the second population of        TILs after 7 days from the start of the rapid expansion; wherein        the second cell culture medium comprises IL-2, OKT-3 (anti-CD3        antibody), and irradiated allogeneic peripheral blood        mononuclear cells (PBMCs); and wherein the rapid expansion is        performed over a period of 14 days or less;    -   (f) harvesting the third population of TILs; and    -   (g) administering a therapeutically effective portion of the        third population of TILs to a patient with the cancer;    -   wherein the cancer is double-refractory metastatic melanoma,        further comprising the step of treating the patient with an IL-2        regimen starting on the day after administration of the third        population of TILs to the patient.

In an embodiment, the invention provides a method of treating a cancerwith a population of tumor infiltrating lymphocytes (TILs) comprisingthe steps of:

-   -   (a) resecting a tumor from a patient, the tumor comprising a        first population of TILs;    -   (b) fragmenting the tumor into tumor fragments;    -   (c) contacting the tumor fragments with a first cell culture        medium;    -   (d) performing an initial expansion of the first population of        TILs in the first cell culture medium to obtain a second        population of TILs, wherein the second population of TILs is at        least 5-fold greater in number than the first population of        TILs, wherein the first cell culture medium comprises IL-2;    -   (e) performing a rapid expansion of the second population of        TILs in a second cell culture medium to obtain a third        population of TILs, wherein the third population of TILs is at        least 50-fold greater in number than the second population of        TILs after 7 days from the start of the rapid expansion; wherein        the second cell culture medium comprises IL-2, OKT-3 (anti-CD3        antibody), and irradiated allogeneic peripheral blood        mononuclear cells (PBMCs); and wherein the rapid expansion is        performed over a period of 14 days or less;    -   (f) harvesting the third population of TILs; and    -   (g) administering a therapeutically effective portion of the        third population of TILs to a patient with the cancer;    -   wherein the cancer is double-refractory metastatic melanoma,        wherein the IL-2 regimen is a high-dose IL-2 regimen comprising        600,000 or 720,000 IU/kg of aldesleukin, or a biosimilar or        variant thereof, administered as a 15-minute bolus intravenous        infusion every eight hours until tolerance.

EXAMPLES

The embodiments encompassed herein are now described with reference tothe following examples. These examples are provided for the purpose ofillustration only and the disclosure encompassed herein should in no waybe construed as being limited to these examples, but rather should beconstrued to encompass any and all variations which become evident as aresult of the teachings provided herein.

Example 1: Processes for the Manufacture of TILs Suitable for Therapy

TILs may be manufactured using methods known in the art and any methoddescribed herein. For example, an exemplary method for expanding TILs isdepicted in FIG. 1. An exemplary timeline for manufacturing TILs andtreating a cancer patient with expanded TILs according to the process ofFIG. 1 is shown in FIG. 2. Surgery (and tumor resection) occurs at thestart, and lymphodepletion chemo refers to non-myeloablativelymphodepletion with chemotherapy as described elsewhere herein.

FIG. 3 illustrates a TIL expansion and therapeutic treatment process,including a “direct to REP” step wherein pre-REP TILs are placeddirectly into a REP process. The total process time is approximately 22days, at which point TILs may be infused into a patient. FIG. 4illustrates a treatment and manufacturing timeline for use with TILsprepared according to the present disclosure and the process of FIG. 3,when the cell count at day 6 is greater than 250×10⁶. In this situation,the TIL product is considered to be very likely to succeed, and the riskof lymphodepleting the patient in anticipation of obtaining suitablefinal TIL product is warranted. FIG. 5 illustrates a treatment andmanufacturing timeline for use with TILs prepared according to thepresent disclosure and the process of FIG. 3, when the cell count at day6 is less than 250×10⁶, and wherein lymphodepletion is begun later so asto allow for an assessment of the viability of the TIL product beforethe decision is made to lymphodeplete the patient. FIG. 6 shows adetailed schematic of a TIL manufacturing process according to FIG. 3.

Example 2: Clinical Study 1 of TIL Therapy in Double-Refractory Melanoma

This Phase 2, multicenter, three-cohort study is designed to assess thesafety and efficacy of a TIL therapy manufactured according to FIG. 1for treatment of patients with metastatic melanoma. Cohorts one and twowill enroll up to 30 patients each and cohort three is a re-treatmentcohort for a second TIL infusion in up to ten patients. The first twocohorts are evaluating two different manufacturing processes for (FIG. 1and FIG. 3, respectively). Patients in cohort one receive fresh,non-cryopreserved TIL (FIG. 1) and cohort two patients receive productmanufactured through a more streamlined and rapid three-week procedure(FIG. 3) yielding a cryopreserved product. The study design is shown inFIG. 7. The study is a Phase 2, multicenter, three cohort study toassess the safety and efficacy of autologous TILs for treatment ofsubpopulations of patients with metastatic melanoma. Key inclusioncriteria include: measurable metastatic melanoma and ≥1 lesionresectable for TIL generation; at least one prior line of systemictherapy; age≥18; and ECOG performance status of 0-1. Treatment cohortsinclude non-cryopreserved TIL product (prepared using the process ofFIG. 1), cryopreserved TIL product (prepared using the process of FIG.3), and retreatment with TIL product for patients without response orwho progress after initial response. The primary endpoint is safety andthe secondary endpoint is efficacy, defined as objective response rate(ORR), complete remission rate (CRR), progression free survival (PFS),duration of response (DOR), and overall survival (OS).

Data from 16 patients in cohort one is presented here. These advancedmetastatic melanoma patients were a median age of 55 and were highlyrefractory to multiple prior lines of therapy with significant tumorburden at baseline. All had prior anti-PD-1 therapy, 88% had anti-CTLA4therapy and 64% had received three or more prior therapies. The resultsshowed that, of the evaluable patients, a 29% objective response ratewas reported including one complete response (CR) continuing beyond 15months post-administration of a single TIL treatment. Furthermore, 77%of patients had reduction in target tumor size. The mean time to firstresponse was 1.6 months, with the CR developing at 6 months. Responseswere observed in patients with tumors carrying wild type or BRAFmutations. The protocol allows for administration of up to 6 doses ofaldesleukin. The median number of aldesleukin administrations was six.

Patient characteristics are shown in FIG. 8 and FIG. 9. The mediannumber of prior therapies was 3 (range: 1-6). The median sum of diameterfor target lesions at baseline was 10.2 cm. 81% of patients had Stage IVdisease. The patient population was highly refractory to multiple priorlines of therapy, with significant tumor burden at baseline, and hadprogressed after at least one checkpoint inhibitor.

Treatment emergent serious adverse events are summarized in FIG. 10, andefficacy results are summarized in FIG. 11. One of 14 patients was notevaluable due to melanoma-related death prior to first tumor assessment.All patients entering the study had received an anti-PD-1 checkpointinhibitor. A waterfall of response plot is shown in FIG. 12. The ORR is29%. Tumor reduction was seen in 77% of patients representing those whohad tumor reduction in the target lesions. Responses were notedregardless of BRAF mutational status including one long lasting CR (15+months). FIG. 13 illustrates time to best response and duration in theclinical study. Mean time to first response was 1.6 months. Medianfollow up for the data shown in FIG. 13 was 4.1 months. FIG. 14illustrates percentage change in sum of diameters in the clinical study.FIG. 15 illustrates scans from a patient in complete remission, showingthe reduction in tumor size.

Additionally, the protocol for this study was amended to both increasethe sample size for the study as well as further define the patientpopulation to patients with unresectable or metastatic melanoma who haveprogressed after immune checkpoint inhibition therapy (e.g., anti-PD-1),and if BRAF mutation-positive, after BRAF targeted therapy.

Based on these results, which illustrate the ability of the TILtherapies of the present disclosure to treat double-refractorymetastatic melanoma, the clinical study has been modified as shown inFIG. 16, and furthermore, the primary endpoint has been changed to ORR,with the secondary endpoints changed to CRR, DCR, PFS, DOR, OR, OS, andsafety.

Example 3: Clinical Study 2 of TIL Therapy in Double-Refractory Melanoma

Alternative processes for TIL production may also be employed in someembodiments, such as the process described in Radvanyi, et al., Clin.Cancer Res. 2012, 18, 6758-70 (including the supporting information),the disclosure of which is incorporated by reference herein. The resultsfrom the use of TILs produced by this method in the treatment ofpatients refractory to both an anti-PD-1 (pembrolizumab or nivolumab)and ipilimumab are shown in FIG. 17.

Example 4: Retrospective Clinical Study

A retrospective study is performed in unresectable, metastatic melanomapatients assessing efficacy data following ≥2 systemic therapies fortheir disease. This is a retrospective chart review study. The studyincludes acquisition of retrospective data on disease response inpatients who are relapsed/refractory unresectable metastatic melanomawho progressed after receiving ≥2 lines of systemic therapies, where thesystemic therapies must include at least one line of PD-1 and BRAFinhibitors for patients with confirmed BRAF mutation positive disease.Selection of patient population is based on prospectively determinedinclusion criteria followed by retrospective chart review.

The primary objective of this retrospective study is to evaluate theobjective response rate (ORR) assessed by the local evaluation followingResponse Evaluation Criteria in Solid Tumors (RECIST) 1.1. Secondaryobjectives of the study include (i) evaluating the efficacy endpoints bythe local evaluation for duration of response (DOR), and disease controlrate (DCR), assessed by RECIST 1.1; (ii) to evaluate overall survival(OS) based on retrospective data of the study population. An exploratoryobjective further includes evaluating the treatment pattern of thisstudy population.

Dose and treatment are based on individual institutional data, with therequirement to have been on a treatment in the second or later line oftreatment for the unresectable, metastatic melanoma.

Retrospective data will be collected from up to 3 large medical databases (e.g., from hospitals, academic institutions, oncologicalcooperative groups) in the United States.

No patients will be actively treated in this retrospective dataevaluation study. A minimum of about 100 patients will be assessed foreligibility for the retrospective data review, based on the availableinstitutional data.

Patients will have relapsed/refractory unresectable, metastatic melanomafollowing ≥2 lines of systemic that must include at least one line ofanti-PD-1 and BRAF inhibitors for patients with confirmed BRAF mutationpositive disease. Patients will be ≥18 years of age at the time ofconsent, and will have an Eastern Cooperative Oncology Group (ECOG)performance status of 0 or 1.

Further criteria for inclusion include; patients must have adequatehematopoietic and organ function; patients have provided writtenauthorization for use and disclosure of protected health information, orthere is institutional regulation allowing use of clinical data, incompliance with GCP and local ethical standards.

Patients meeting the following criteria will be excluded from the study:patients with melanoma of uveal/ocular origin; patients with symptomaticand/or untreated brain metastases (of any size and any number); patientswho have had another primary malignancy within the previous 3 years(with the exception of carcinoma in situ of the breast, cervix, orbladder, localized prostate cancer and nonmelanoma skin cancer that hasbeen adequately treated); patients who have been shown to be BRAFmutation positive (V600), but have not received prior systemic therapywith a BRAF-directed kinase inhibitor.

Efficacy will be assessed based on the application of RECIST 1.1 to thedata available in the medical charts of the patients identifiedaccording to the inclusion/exclusion criteria. The following parameterswill be calculated: ORR, DOR, DCR. Data will be reported by individualinstitutional data and as aggregate, if feasible.

OS summary will be also assessed pending available individualinstitutional data. If feasible, aggregate OS data will be reported.

The primary statistical analysis is based on the efficacy parametersobtained from the retrospective data from each institution and it willbe performed by individual set of retrospective data per institution.

Statistical comparison among retrospective data sets may or may not beperformed.

Patients meeting RECIST 1.1 criteria for a confirmed complete (CR) orpartial (PR) response will be classified as responders in the analysisof the ORR.

All time-to-event efficacy endpoints will use the Kaplan-Meier method tosummarize the data. The time origin for all such analyses (except forresponse duration) will be the date on which patients began treatmentwith the study therapy.

There may or may not be formal comparisons among individualretrospective data sets.

Example 5: Preparation of Media for Pre-REP and REP Processes

This Example describes the procedure for the preparation of tissueculture media for use in protocols involving the culture of tumorinfiltrating lymphocytes (TIL) derived from various tumor typesincluding, but not limited to, metastatic melanoma, head and necksquamous cell carcinoma (HNSCC), ovarian carcinoma, triple-negativebreast carcinoma, and lung adenocarcinoma. This media can be used forpreparation of any of the TILs described in the present application andExamples.

Preparation of CM1

Removed the following reagents from cold storage and warmed them in a37° C. water bath: (RPMI1640, Human AB serum, 200 mM L-glutamine).Prepared CM1 medium according to Table 19 below by adding each of theingredients into the top section of a 0.2 μm filter unit appropriate tothe volume to be filtered. Store at 4° C.

TABLE 19 Preparation of CM1 Final Final Final Ingredient concentrationVolume 500 ml Volume IL RPMI1640 NA 450 ml 900 ml  Human AB serum, 50 ml100 ml heat-inactivated 10% 200 mM L-glutamine  2 mM  5 ml 10 ml  55 mMBME 55 μM  0.5 ml 1 ml 50 mg/ml gentamicin 50 μg/ml  0.5 ml 1 ml sulfate

On the day of use, prewarmed required amount of CM1 in 37° C. water bathand add 6000 IU/ml IL-2.

Additional supplementation—as needed according to Table 20.

TABLE 20 Additional supplementation of CM1, as needed. Supplement Stockconcentration Dilution Final concentration GlutaMAXTm 200 mM 1:100   2mM Penicillin/ 10,000 U/ml penicillin 1:100 100 U/ml penicillinstreptomycin 10,000 μg/ml 100 μg/ml streptomycin streptomycinAmphotericin B 250 μg/ml 1:100 2.5 μg/ml

Preparation of CM2

Removed prepared CM1 from refrigerator or prepare fresh CM1 as perSection 7.3 above. Removed AIM-V® from refrigerator and prepared theamount of CM2 needed by mixing prepared CM1 with an equal volume ofAIM-V® in a sterile media bottle. Added 3000 IU/ml IL-2 to CM2 medium onthe day of usage. Made sufficient amount of CM2 with 3000 IU/ml IL-2 onthe day of usage. Labeled the CM2 media bottle with its name, theinitials of the preparer, the date it was filtered/prepared, thetwo-week expiration date and store at 4° C. until needed for tissueculture.

Preparation of CM3

Prepared CM3 on the day it was required for use. CM3 was the same asAIM-V® medium, supplemented with 3000 IU/ml IL-2 on the day of use.Prepared an amount of CM3 sufficient to experimental needs by addingIL-2 stock solution directly to the bottle or bag of AIM-V. Mixed wellby gentle shaking. Label bottle with “3000 IU/ml IL-2” immediately afteradding to the AIM-V. If there was excess CM3, stored it in bottles at 4°C. labeled with the media name, the initials of the preparer, the datethe media was prepared, and its expiration date (7 days afterpreparation). Discarded media supplemented with IL-2 after 7 daysstorage at 4° C.

Preparation of CM4

CM4 was the same as CM3, with the additional supplement of 2 mMGlutaMAX™ (final concentration). For every 1 L of CM3, added 10 ml of200 mM GlutaMAX™. Prepared an amount of CM4 sufficient to experimentalneeds by adding IL-2 stock solution and GlutaMAX™ stock solutiondirectly to the bottle or bag of AIM-V. Mixed well by gentle shaking.Labeled bottle with “3000 IL/nil IL-2 and GlutaMAX” immediately afteradding to the AIM-V. If there was excess CM4, stored it in bottles at 4°C. labeled with the media name, “GlutaMAX”, and its expiration date (7days after preparation). Discarded media supplemented with IL-2 after7-days storage at 4° C.

Example 6: Use of IL-2, IL-15, and IL-21 Cytokine Cocktail

This example describes the use of IL-2, IL-15, and IL-21 cytokines,which serve as additional T cell growth factors, in combination with theTIL process of Examples 1 to 10.

Using the process of Examples 1 to 10, TILs were grown from colorectal,melanoma, cervical, triple negative breast, lung and renal tumors inpresence of IL-2 in one arm of the experiment and, in place of IL-2, acombination of IL-2, IL-15, and IL-21 in another arm at the initiationof culture. At the completion of the pre-REP, cultures were assessed forexpansion, phenotype, function (CD107a+ and IFN-γ) and TCR Vβrepertoire. IL-15 and IL-21 are described elsewhere herein and inGruijl, et al., IL-21 promotes the expansion of CD27+CD28+ tumorinfiltrating lymphocytes with high cytotoxic potential and lowcollateral expansion of regulatory T cells, Santegoets, S. J., J TranslMed., 2013, 11:37(https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3626797/).

The results showed that enhanced TIL expansion (>20%), in both CD4⁺ andCD8⁺ cells in the IL-2, IL-15, and IL-21 treated conditions wereobserved in multiple histologies relative to the IL-2 only conditions.There was a skewing towards a predominantly CD8⁺ population with askewed TCR Vβ repertoire in the TILs obtained from the IL-2, IL-15, andIL-21 treated cultures relative to the IL-2 only cultures. IFN-γ andCD107a were elevated in the IL-2, IL-15, and IL-21 treated TILs, incomparison to TILs treated only IL-2.

Example 7: Qualifying Individual Lots of Gamma-Irradiated PeripheralMononuclear Cells

This Example describes a novel abbreviated procedure for qualifyingindividual lots of gamma-irradiated peripheral mononuclear cells (PBMCs,also known as MNC) for use as allogeneic feeder cells in the exemplarymethods described herein.

Each irradiated MNC feeder lot was prepared from an individual donor.Each lot or donor was screened individually for its ability to expandTIL in the REP in the presence of purified anti-CD3 (clone OKT3)antibody and interleukin-2 (IL-2). In addition, each lot of feeder cellswas tested without the addition of TIL. to verify that the received doseof gamma radiation was sufficient to render them replicationincompetent.

BACKGROUND

Gamma-irradiated, growth-arrested MNC feeder cells were required for REPof TIL. Membrane receptors on the feeder MNCs bind to anti-CD3 (cloneOKT3) antibody and crosslink to TIL in the REP flask, stimulating theTIL to expand. Feeder lots were prepared from the leukapheresis of wholeblood taken from individual donors. The leukapheresis product wassubjected to centrifugation over Ficoll-Hypaque, washed, irradiated, andcryopreserved under GMP conditions.

It is important that patients who received TIL therapy not be infusedwith viable feeder cells as this can result in Graft-Versus-Host Disease(GVHD). Feeder cells are therefore growth-arrested by dosing the cellswith gamma-irradiation, resulting in double strand DNA breaks and theloss of cell viability of the MNC cells upon reculture.

Evaluation Criteria and Experimental Set-Up

Feeder lots were evaluated on two criteria: 1) their ability to expandTIL in co-culture >100-fold and 2) their replication incompetency.

Feeder lots were tested in mini-REP format utilizing two primary pre-REPTIL lines grown in upright T25 tissue culture flasks. Feeder lots weretested against two distinct TIL lines, as each TIL line is unique in itsability to proliferate in response to activation in a REP. As a control,a lot of irradiated MNC feeder cells which has historically been shownto meet the criteria above was run alongside the test lots.

To ensure that all lots tested in a single experiment receive equivalenttesting, sufficient stocks of the same pre-REP TIL lines were availableto test all conditions and all feeder lots.

For each lot of feeder cells tested, there was a total of six T25flasks: Pre-REP TIL line #1 (2 flasks); Pre-REP TIL line #2 (2 flasks);and Feeder control (2 flasks). Flasks containing TIL lines #1 and #2evaluated the ability of the feeder lot to expand TIL. The feedercontrol flasks evaluated the replication incompetence of the feeder lot.

EXPERIMENTAL PROTOCOL Day −2/3, Thaw of TIL Lines

Prepared CM2 medium. Warmed CM2 in 37° C. water bath. Prepared 40 ml ofCM2 supplemented with 3000 IU/ml IL-2. Keep warm until use. Placed 20 mlof pre-warmed CM2 without IL-2 into each of two 50 ml conical tubeslabeled with names of the TIL lines used. Removed the two designatedpre-REP TIL lines from LN2 storage and transferred the vials to thetissue culture room. Thawed vials by placing them inside a sealed zipperstorage bag in a 37° C. water bath until a small amount of ice remains.

Using a sterile transfer pipet, immediately transferred the contents ofvial into the 20 ml of CM2 in the prepared, labeled 50 ml conical tube.QS to 40 ml using CM2 without IL-2 to wash cells. Centrifuged at 400×CFfor 5 minutes. Aspirated the supernatant and resuspend in 5 ml warm CM2supplemented with 3000 IU/ml IL-2.

Removed small aliquot (20 μl) in duplicate for cell counting using anautomated cell counter. Record the counts. While counting, placed the 50ml conical tube with TIL cells into a humidified 37° C., 5% CO₂incubator, with the cap loosened to allow for gas exchange. Determinedcell concentration and diluted TIL to 1×10⁶ cells/ml in CM2 supplementedwith IL-2 at 3000 IU/ml.

Cultured in 2 ml/well of a 24-well tissue culture plate in as many wellsas needed in a humidified 37° C. incubator until Day 0 of the mini-REP.Cultured the different TIL lines in separate 24-well tissue cultureplates to avoid confusion and potential cross-contamination.

Day 0, Initiate Mini-REP

Prepared enough CM2 medium for the number of feeder lots to be tested.(e.g., for testing 4 feeder lots at one time, prepared 800 ml of CM2medium). Aliquotted a portion of the CM2 prepared above and supplementedit with 3000 IU/ml IL-2 for the culturing of the cells. (e.g., fortesting 4 feeder lots at one time, prepare 500 ml of CM2 medium with3000 IU/ml IL-2).

Working with each TIL line separately to prevent cross-contamination,removed the 24-well plate with TIL culture from the incubator andtransferred to the BSC.

Using a sterile transfer pipet or 100-1000 μl Pipettor and tip, removedabout 1 ml of medium from each well of TIL to be used and place in anunused well of the 24-well tissue culture plate.

Using a fresh sterile transfer pipet or 100-1000 μl Pipettor and tip,mixed remaining medium with TIL in wells to resuspend the cells and thentransferred the cell suspension to a 50 ml conical tube labeled with theTIL name and recorded the volume.

Washed the wells with the reserved media and transferred that volume tothe same 50 ml conical tube. Spun the cells at 400×CF to collect thecell pellet. Aspirated off the media supernatant and resuspend the cellpellet in 2-5 ml of CM2 medium containing 3000 IU/ml IL-2, volume to beused based on the number of wells harvested and the size of thepellet—volume should be sufficient to ensure a concentration of >1.3×10⁶cells/ml.

Using a serological pipet, mixed the cell suspension thoroughly andrecorded the volume. Removed 200 μl for a cell count using an automatedcell counter. While counting, placed the 50 ml conical tube with TILcells into a humidified, 5% CO₂, 37° C. incubator, with the cap loosenedto allow gas exchange. Recorded the counts.

Removed the 50 ml conical tube containing the TIL cells from theincubator and resuspend them cells at a concentration of 1.3×10⁶cells/ml in warm CM2 supplemented with 3000 IU/ml IL-2. Returned the 50ml conical tube to the incubator with a loosened cap.

Repeated steps above for the second TIL line.

Just prior to plating the TIL into the T25 flasks for the experiment,TIL were diluted 1:10 for a final concentration of 1.3×10⁵ cells/ml asper below.

Prepare MACS GMP CD3 Pure (OKT3) Working Solution

Took out stock solution of OKT3 (1 mg/ml) from 4° C. refrigerator andplaced in BSC. A final concentration of 30 ng/ml OKT3 was used in themedia of the mini-REP.

600 ng of OKT3 were needed for 20 ml in each T25 flask of theexperiment; this was the equivalent of 60 μl of a 10 μg/ml solution foreach 20 ml, or 360.1 for all 6 flasks tested for each feeder lot.

For each feeder lot tested, made 400 μl of a 1:100 dilution of 1 mg/mlOKT3 for a working concentration of 10 μg/ml (e.g., for testing 4 feederlots at one time, make 1600.1 of a 1:100 dilution of 1 mg/ml OKT3: 16 μlof 1 mg/ml OKT3+1.584 ml of CM2 medium with 3000 IU/ml IL-2.)

Prepare T25 Flasks

Labeled each flask and filled flask with the CM2 medium prior topreparing the feeder cells. Placed flasks into 37° C. humidified 5% CO₂incubator to keep media warm while waiting to add the remainingcomponents. Once feeder cells were prepared, the components will beadded to the CM2 in each flask.

TABLE 21 Solutions Volume in Volume in control co-culture (feederComponent flasks only) flasks CM2 + 3000 IU/ml IL-2 18 ml 19 ml MNC: 1.3× 10⁷/ml in CM2 + 3000IU IL-2  1 ml  1 ml (final concentration 1/.3 ×10⁷/flask) OKT3: 10 μg/ml in CM2 + 3000IU of IL-2 60 μl 60 μl TIL: 1.3 ×10⁵/ml in CM2 with 3000IU of IL-2  1 ml 0 (final concentration 1.3 ×10⁵/flask)

Prepare Feeder Cells

A minimum of 78×10⁶ feeder cells were needed per lot tested for thisprotocol. Each 1 ml vial frozen by SDBB had 100×10⁶ viable cells uponfreezing. Assuming a 50% recovery upon thaw from LN2 storage, it wasrecommended to thaw at least two 1 ml vials of feeder cells per lotgiving an estimated 100×10⁶ viable cells for each REP. Alternately, ifsupplied in 1.8 ml vials, only one vial provided enough feeder cells.

Before thawing feeder cells, pre-warmed approximately 50 ml of CM2without IL-2 for each feeder lot to be tested. Removed the designatedfeeder lot vials from LN2 storage, placed in zipper storage bag, andplace on ice. Thawed vials inside closed zipper storage bag by immersingin a 37° C. water bath. Removed vials from zipper bag, spray or wipewith 70% EtOH and transferred vials to BSC.

Using a transfer pipet immediately transferred the contents of feedervials into 30 ml of warm CM2 in a 50 ml conical tube. Washed vial with asmall volume of CM2 to remove any residual cells in the vial.Centrifuged at 400×CF for 5 minutes. Aspirated the supernatant andresuspended in 4 ml warm CM2 plus 3000 IU/ml IL-2. Removed 200 μl forcell counting using the Automated Cell Counter. Recorded the counts.

Resuspended cells at 1.3×10⁷ cells/ml in warm CM2 plus 3000 IU/ml IL-2.Diluted TIL cells from 1.3×10⁶ cells/ml to 1.3×10⁵ cells/ml.

Setup Co-Culture

Diluted TIL cells from 1.3×10⁶ cells/ml to 1.3×10⁵ cells/ml. Added 4.5ml of CM2 medium to a 15 ml conical tube. Removed TIL cells fromincubator and resuspended well using a 10 ml serological pipet. Removed0.5 ml of cells from the 1.3×10⁶ cells/ml TIL suspension and added tothe 4.5 ml of medium in the 15 ml conical tube. Returned TIL stock vialto incubator. Mixed well. Repeated for the second TIL line.

Transferred flasks with pre-warmed media for a single feeder lot fromthe incubator to the BSC. Mixed feeder cells by pipetting up and downseveral times with a 1 ml pipet tip and transferred 1 ml (1.3×10⁷ cells)to each flask for that feeder lot. Added 60 μl of OKT3 working stock (10μg/ml) to each flask. Returned the two control flasks to the incubator.

Transferred 1 ml (1.3×10⁵) of each TIL lot to the correspondinglylabeled T25 flask. Returned flasks to the incubator and incubateupright. Did not disturb until Day 5.

Repeated for all feeder lots tested.

Day 5, Media Change

Prepared CM2 with 3000 IU/ml IL-2. 10 ml is needed for each flask. Witha 10 ml pipette, transferred 10 ml warm CM2 with 3000 IU/ml IL-2 to eachflask. Returned flasks to the incubator and incubated upright until Day7. Repeated for all feeder lots tested.

Day 7, Harvest

Removed flasks from the incubator and transfer to the BSC, care as takennot to disturb the cell layer on the bottom of the flask. Withoutdisturbing the cells growing on the bottom of the flasks, removed 10 mlof medium from each test flask and 15 ml of medium from each of thecontrol flasks.

Using a 10 ml serological pipet, resuspended the cells in the remainingmedium and mix well to break up any clumps of cells. After thoroughlymixing cell suspension by pipetting, removed 200 μl for cell counting.Counted the TIL using the appropriate standard operating procedure inconjunction with the automatic cell counter equipment. Recorded countsin Day 7.

Repeated for all feeder lots tested.

Feeder control flasks were evaluated for replication incompetence andflasks containing TIL were evaluated for fold expansion from Day 0according to Table 22 below.

Day 7, Continuation of Feeder Control Flasks to Day 14

After completing the Day 7 counts of the feeder control flasks, added 15ml of fresh CM2 medium containing 3000 IU/ml IL-2 to each of the controlflasks. Returned the control flasks to the incubator and incubated in anupright position until Day 14.

Day 14, Extended Non-Proliferation of Feeder Control Flasks

Removed flasks from the incubator and transfer to the BSC, care wastaken not to disturb the cell layer on the bottom of the flask. Withoutdisturbing the cells growing on the bottom of the flasks, removedapproximately 17 ml of medium from each control flasks. Using a 5 mlserological pipet, resuspended the cells in the remaining medium andmixed well to break up any clumps of cells. Recorded the volumes foreach flask.

After thoroughly mixing cell suspension by pipetting, removed 200 μl forcell counting. Counted the TIL using the appropriate standard operatingprocedure in conjunction with the automatic cell counter equipment.Recorded counts.

Repeated for all feeder lots tested.

Results and Acceptance Criteria Results

The dose of gamma irradiation was sufficient to render the feeder cellsreplication incompetent. All lots were expected to meet the evaluationcriteria and also demonstrated a reduction in the total viable number offeeder cells remaining on Day 7 of the REP culture compared to Day 0.

All feeder lots were expected to meet the evaluation criteria of100-fold expansion of TIL growth by Day 7 of the REP culture.

Day 14 counts of Feeder Control flasks were expected to continue thenon-proliferative trend seen on Day 7.

Acceptance Criteria

The following acceptance criteria were met for each replicate TIL linetested for each lot of feeder cells

Acceptance was two-fold, as follows (outlined in Table 22 below).

TABLE 22 Acceptance Criteria Test Acceptance criteria Irradiation of Nogrowth observed at 7 and 14 days MNC/Replication Incompetence TILexpansion At least a 100-fold expansion of each TIL (minimum of 1.3 ×10⁷ viable cells)

Evaluated whether the dose of radiation was sufficient to render the MNCfeeder cells replication incompetent when cultured in the presence of 30ng/ml OKT3 antibody and 3000 IU/ml IL-2. Replication incompetence wasevaluated by total viable cell count (TVC) as determined by automatedcell counting on Day 7 and Day 14 of the REP.

Acceptance criteria was “No Growth,” meaning the total viable cellnumber has not increased on Day 7 and Day 14 from the initial viablecell number put into culture on Day 0 of the REP.

Evaluated the ability of the feeder cells to support TIL expansion. TILgrowth was measured in terms of fold expansion of viable cells from theonset of culture on Day 0 of the REP to Day 7 of the REP. On Day 7, TILcultures achieved a minimum of 100-fold expansion, (i.e., greater than100 times the number of total viable TIL cells put into culture on REPDay 0), as evaluated by automated cell counting.

Contingency Testing of MNC Feeder Lots that do not Meet AcceptanceCriteria

In the event that an MNC feeder lot did not meet the either of theacceptance criteria outlined above, the following steps will be taken toretest the lot to rule out simple experimenter error as its cause.

If there are two or more remaining satellite testing vials of the lot,then the lot was retested. If there were one or no remaining satellitetesting vials of the lot, then the lot was failed according to theacceptance criteria listed above.

In order to be qualified, the lot in question and the control lot had toachieve the acceptance criteria above. Upon meeting these criteria, thelot was then released for use.

Example 8: Qualifying Individual Lots of Gamma-Irradiated PeripheralBlood Mononuclear Cells

This Example describes a novel abbreviated procedure for qualifyingindividual lots of gamma-irradiated peripheral blood mononuclear cells(PBMC) for use as allogeneic feeder cells in the exemplary methodsdescribed herein. This example provides a protocol for the evaluation ofirradiated PBMC cell lots for use in the production of clinical lots ofTIL. Each irradiated PBMC lot was prepared from an individual donor.Over the course of more than 100 qualification protocols, it was beenshown that, in all cases, irradiated PBMC lots from SDBB (San DiegoBlood Bank) expand TIL >100-fold on Day 7 of a REP. This modifiedqualification protocol was intended to apply to irradiated donor PBMClots from SDBB which were then further tested to verify that thereceived dose of gamma radiation was sufficient to render themreplication incompetent. Once demonstrated that they maintainedreplication incompetence over the course of 14 days, donor PBMC lotswere considered “qualified” for usage to produce clinical lots of TIL.

Background

Gamma-irradiated, growth-arrested PBMC were required for currentstandard REP of TIL. Membrane receptors on the PBMCs bind to anti-CD3(clone OKT3) antibody and crosslink to TIL in culture, stimulating theTIL to expand. PBMC lots were prepared from the leukapheresis of wholeblood taken from individual donors. The leukapheresis product wassubjected to centrifugation over Ficoll-Hypaque, washed, irradiated, andcryopreserved under GMP conditions.

It is important that patients who received TIL therapy not be infusedwith viable PBMCs as this could result in Graft-Versus-Host Disease(GVHD). Donor PBMCs are therefore growth-arrested by dosing the cellswith gamma-irradiation, resulting in double strand DNA breaks and theloss of cell viability of the PBMCs upon reculture.

Evaluation Criteria

7.2.1 Evaluation criterion for irradiated PBMC lots was theirreplication incompetency.

Experimental Set-Up

Feeder lots were tested in mini-REP format as if they were to beco-cultured with TIL, using upright T25 tissue culture flasks. Controllot: One lot of irradiated PBMCs, which had historically been shown tomeet the criterion of 7.2.1, was run alongside the experimental lots asa control. For each lot of irradiated donor PBMC tested, duplicateflasks were run.

Experimental Protocol Day 0

Prepared ˜90 ml of CM2 medium for each lot of donor PBMC to be tested.Kept CM2 warm in 37° C. water bath. Thawed an aliquot of 6×10⁶ IU/mlIL-2. Returned the CM2 medium to the BSC, wiping with 70% EtOH prior toplacing in hood. For each lot of PBMC tested, removed about 60 ml of CM2to a separate sterile bottle. Added IL-2 from the thawed 6×10⁶ IU/mlstock solution to this medium for a final concentration of 3000 IU/ml.Labeled this bottle as “CM2/IL2” (or similar) to distinguish it from theunsupplemented CM2.

Prepare OKT3

Took out the stock solution of anti-CD3 (OKT3) from the 4° C.refrigerator and placed in the BSC. A final concentration of 30 ng/mlOKT3 was used in the media of the mini-REP. Prepared a 10 μg/ml workingsolution of anti-CD3 (OKT3) from the 1 mg/ml stock solution. Placed inrefrigerator until needed.

For each PBMC lot tested, prepare 150 μl of a 1:100 dilution of theanti-CD3 (OKT3) stock. For example, for testing 4 PBMC lots at one time,prepare 600 μl of 10 μg/ml anti-CD3 (OKT3) by adding 6 μl of the 1 mg/mlstock solution to 5941l of CM2 supplemented with 3000 IU/ml IL-2.

Prepare Flasks

7.4.8 Added 19 ml per flask of CM2/IL-2 to the labeled T25 flasks andplaced flasks into 37° C., humidified, 5% CO₂ incubator while preparingcells.

Prepare Irradiate PBMC

Retrieved vials of PBMC lots to be tested from LN2 storage. These wereplaced at −80° C. or kept on dry ice prior to thawing. Placed 30 ml ofCM2 (without IL-2 supplement) into 50 ml conical tubes for each lot tobe thawed. Labeled each tube with the different lot numbers of the PBMCto be thawed. Capped tubes tightly and place in 37° C. water bath priorto use. As needed, returned 50 ml conical tubes to the BSC, wiping with70% EtOH prior to placing in the hood.

Removed a vial PBMC from cold storage and place in a floating tube rackin a 37° C. water bath to thaw. Allowed thaw to proceed until a smallamount of ice remains in the vial. Using a sterile transfer pipet,immediately transferred the contents of the vial into the 30 ml of CM2in the 50 ml conical tube. Removed about 1 ml of medium from the tube torinse the vial; returned rinse to the 50 ml conical tube. Capped tightlyand swirl gently to wash cells.

Centrifuged at 400×g for 5 min at room temperature. Aspirated thesupernatant and resuspend the cell pellet in 1 ml of warm CM2/IL-2 usinga 1000 μl pipet tip. Alternately, prior to adding medium, resuspendedcell pellet by dragging capped tube along an empty tube rack. Afterresuspending the cell pellet, brought volume to 4 ml using CM2/IL-2medium. Recorded volume.

Removed a small aliquot (e.g., 100 μl) for cell counting using anautomated cell counter. Performed counts in duplicate according to theparticular automated cell counter SOP. It most likely was necessary toperform a dilution of the PBMC prior to performing the cell counts. Arecommended starting dilution was 1:10, but this varied depending on thetype of cell counter used. Recorded the counts.

Adjusted concentration of PBMC to 1.3×10⁷ cells/ml using CM2/IL-2medium. Mixed well by gentle swirling or by gently aspiratingup-and-down using a serological pipet.

Set Up Culture Flasks

Returned two labeled T25 flasks to the BSC from the tissue cultureincubator. Returned the 10 μg/ml vial of anti-CD3/OKT3 to the BSC. Added1 ml of the 1.3×10⁷ PBMC cell suspension to each flask. Added 60 μl ofthe 10 μg/ml anti-CD3/OKT3 to each flask. Returned capped flasks to thetissue culture incubators for 14 days of growth without disturbance.Placed anti-CD3/OKT3 vial back into the refrigerator until needed forthe next lot. Repeated for each lot of PBMC to be evaluated.

Day 14, Measurement of Non-Proliferation of PBMC

Returned the duplicate T25 flasks to the BSC. For each flask, using afresh 10 ml serological pipet, removed −17 ml from each of the flasks,then carefully pulled up the remaining media to measure the volumeremaining in the flasks. Recorded volume.

Mixed sample well by pipetting up and down using the same serologicalpipet.

Removed a 200 μl sample from each flask for counting. Counted cellsusing an automated cell counter. Repeated steps 7.4.26-7.4.31 for eachlot of PBMC being evaluated.

Results and Acceptance Criterion Results

The dose of gamma irradiation was expected to be sufficient to renderthe feeder cells replication incompetent. All lots were expected to meetthe evaluation criterion, demonstrating a reduction in the total viablenumber of feeder cells remaining on Day 14 of the REP culture comparedto Day 0.

Acceptance Criterion: The following acceptance criterion were met foreach irradiated donor PBMC lot tested: “No growth”—meant that the totalnumber of viable cells on Day 14 was less than the initial viable cellnumber put into culture on Day 0 of the REP.

Contingency Testing of PBMC Lots which do not Meet Acceptance Criterion.

In the event than an irradiated donor PBMC lot did not meet theacceptance criterion above, the following steps were taken to retest thelot to rule out simple experimenter error as the cause of its failure.If there were two or more remaining satellite vials of the lot, then thelot was retested. If there are one or no remaining satellite vials ofthe lot, then the lot was failed according to the acceptance criterionabove.

To be qualified, a PBMC lot going through contingency testing had boththe control lot and both replicates of the lot in question achieve theacceptance criterion. Upon meeting this criterion, the lot was thenreleased for use.

Example 9: Preparation of 11-2 Stock Solution (Cellgenix)

This Example describes the process of dissolving purified, lyophilizedrecombinant human interleukin-2 into stock samples suitable for use infurther tissue culture protocols, including all of those described inthe present application and Examples, including those that involve usingrhIL-2.

Procedure

Prepared 0.2% Acetic Acid solution (HAc). Transferred 29 mL sterilewater to a 50 mL conical tube. Added 1 mL IN acetic acid to the 50 mLconical tube. Mixed well by inverting tube 2-3 times. Sterilized the HAcsolution by filtration using a Steriflip filter

Prepare 1% HSA in PBS. Added 4 mL of 25% HSA stock solution to 96 mL PBSin a 150 mL sterile filter unit. Filtered solution. Stored at 4° C. Foreach vial of rhIL-2 prepared, fill out forms.

Prepared rhIL-2 stock solution (6×10⁶ IU/mL final concentration). Eachlot of rh1 L-2 was different and required information found in themanufacturer's Certificate of Analysis (COA), such as: 1) Mass of rhIL-2per vial (mg), 2) Specific activity of rhIL-2 (IU/mg) and 3) Recommended0.2% HAc reconstitution volume (mL).

Calculated the volume of 1% HSA required for rhIL-2 lot by using theequation below:

${\left( \frac{{Vial}\mspace{14mu} {Mass}\mspace{14mu} ({mg}) \times {Biological}\mspace{14mu} {Activity}\mspace{14mu} \left( \frac{IU}{mg} \right)}{6 \times 10^{6}\mspace{11mu} \frac{IU}{mL}} \right) - {{HAc}\mspace{14mu} {vol}\mspace{14mu} ({mL})}} = {1\% \mspace{14mu} {HSA}\mspace{14mu} {vol}\mspace{14mu} ({mL})}$${\left( \frac{1\mspace{14mu} {mg} \times 25 \times 10^{6}\frac{IU}{mg}}{6 \times 10^{6}\frac{IU}{mL}} \right) - {2\mspace{14mu} {mL}}} = {2.167\mspace{14mu} {mL}\mspace{14mu} {HSA}}$

For example, according to CellGenix's rhIL-2 lot 10200121 COA, thespecific activity for the 1 mg vial is 25×10⁶ 1 U/mg. It recommendsreconstituting the rhIL-2 in 2 mL 0.2% HAc.

Wiped rubber stopper of IL-2 vial with alcohol wipe. Using a 16G needleattached to a 3 mL syringe, injected recommended volume of 0.2% HAc intovial. Took care to not dislodge the stopper as the needle is withdrawn.Inverted vial 3 times and swirled until all powder is dissolved.Carefully removed the stopper and set aside on an alcohol wipe. Addedthe calculated volume of 1% HSA to the vial.

Storage of rhIL-2 solution. For short-term storage (<72 hrs), storedvial at 4° C. For long-term storage (>72 hrs), aliquotted vial intosmaller volumes and stored in cryovials at −20° C. until ready to use.Avoided freeze/thaw cycles. Expired 6 months after date of preparation.Rh-IL-2 labels included vendor and catalog number, lot number,expiration date, operator initials, concentration and volume of aliquot.

Example 10: Cryopreservation Process

This example describes the cryopreservation process method for TILsprepared with the abbreviated, closed procedure described above inExample 8 using the CryoMed Controlled Rate Freezer, Model 7454 (ThermoScientific).

The equipment used, in addition to that described in Example 9, is asfollows: aluminum cassette holder rack (compatible with CS750 freezerbags), cryostorage cassettes for 750 mL bags, low pressure (22 psi)liquid nitrogen tank, refrigerator, thermocouple sensor (ribbon type forbags), and CryoStore CS750 Freezing bags (OriGen Scientific).

The freezing process provides for a 0.5° C. rate from nucleation to −20°C. and 1° C. per minute cooling rate to −80° C. end temperature. Theprogram parameters are as follows: Step 1—wait at 4° C.; Step 2: 1.0°C./min (sample temperature) to −4° C.; Step 3: 20.0° C./min (chambertemperature) to −45° C.; Step 4: 10.0° C./min (chamber temperature) to−10.0° C.; Step 5: 0.5° C./min (chamber temperature) to −20° C.; andStep 6: 1.0° C./min (sample temperature) to −80° C.

Example 11: Production of a Cryopreserved TIL Cell Therapy Using aClosed System

This examples describes the cGMP manufacture of Iovance Biotherapeutics,Inc.

TIL Cell Therapy Process in G-Rex Flasks according to current GoodTissue Practices and current Good Manufacturing Practices. This materialwill be manufactured under US FDA Good Manufacturing PracticesRegulations (21 CFR Part 210, 211, 1270, and 1271), and applicable ICHQ7 standards for Phase I through Commercial Material.

The process summary is provided in Table 23 below.

TABLE 23 Process summary Estimated Estimated Day Total (post-Anticipated Volume seed) Activity Target Criteria Vessels (mL) 0 Tumor≤50 desirable G-Rex100MCS ≤1000 Dissection tumor 1 flask fragments perG- Rex100MCS 11 REP Seed 5-200 × 10⁶ G-Rex500MCS ≤5000 viable cells 1flasks per G- Rex500MCS 16 REP Split 1 × 10⁹ G-Rex500MCS ≤ 5 ≤25000viable cells flasks per G- Rex500MCS 22 Harvest Total 3-4 CS-750 bags≤530 available cells

Throughout this Example, assume 1.0 mL/L=1.0 g/kg, unless otherwisespecified. Once opened, the following expires apply at 2° C. −8° C.:Human Serum, type AB (HI) Gemini, 1 month; 2-mercaptoethanol, 1 month.Gentamicin Sulfate, 50 mg/ml stock may be kept at room temperature for 1month. Bags containing 10 L of AIM-V media may be warmed at roomtemperature once only for up to 24 hours prior to use. During the Day 22harvest two Gatherex™ may be used to harvest the TIL from theG-Rex500MCS flasks.

Day 0 CM1 Media Preparation

Prepared RPMI 1640 Media. In the BSC, using an appropriately sizedpipette, removed 100.0 mL from 1000 mL RPMI 1640 Media and placed intoan appropriately sized container labeled “Waste”.

In the BSC added reagents to RPMI 1640 Media bottle. Added the followingreagents to the RPMI 1640 Media bottle as shown in table. Recordedvolumes added. Amount Added per bottle: Heat Inactivated Human AB Serum(100.0 mL); GlutaMax (10.0 mL); Gentamicin sulfate, 50 mg/mL (1.0 mL);2-mercaptoethanol (1.0 mL)

Capped RPMI 1640 Media bottle and swirled bottle to ensure reagents weremixed thoroughly. Filtered RPMI 1640 Media from Step 8.1.6 through 1 L0.22-micron filter unit. Labeled filtered media. Aseptically capped thefiltered media and labeled with the following information.

Thawed one 1.1 mL IL-2 aliquot (6×10⁶ IU/mL) (BR71424) until all ice hadmelted. Recorded IL-2: Lot # and Expiry. Transferred IL-2 stock solutionto media. In the BSC, transferred 1.0 mL of IL-2 stock solution to theCM1 Day 0 Media Bottle prepared in Step 8.1.8. Added CM1 Day 0 Media 1bottle and IL-2 (6×10⁶ IU/mL) 1.0 mL. Capped and swirled the bottle tomix media containing IL-2. Relabeled as “Complete CM1 Day 0 Media”.

Removed 20.0 mL of media using an appropriately sized pipette anddispensed into a 50 mL conical tube. In BSC, transferred 25.0 mL of“Complete CM1 Day 0 Media” (prepared in Step 8.1.13) to a 50 mL conicaltube. Labeled the tube as “Tissue Pieces”. Aseptically passedG-Rex100MCS (W3013130) into the BSC. In the BSC, closed all clamps onthe G-Rex100MCS, leaving vent filter clamp open. Connected the red lineof G-Rex100MCS flask to the larger diameter end of the repeater pumpfluid transfer set (W3009497) via luer connection. Staged Baxa pump nextto BSC. Removed pump tubing section of repeater pump fluid transfer setfrom BSC and installed in repeater pump. Within the BSC, removed thesyringe from Pumpmatic Liquid-Dispensing System (PLDS) (W3012720) anddiscarded.

Connected PLDS pipette to the smaller diameter end of repeater pumpfluid transfer set via luer connection and placed pipette tip in“Complete CM1 Day 0 Media” for aspiration. Opened all clamps betweenmedia and G-Rex100MCS. Pumped Complete CM1 media into G-Rex100MCS flask.Set the pump speed to “High” and “9” and pumped all Complete CM1 Day 0Media into G-Rex100MCS flask. Once all media was transferred, clearedthe line and stopped pump.

Disconnected pump from flask. Ensured all clamps were closed on theflask, except vent filter. Removed the repeater pump fluid transfer setfrom the red media line, and placed a red cap (W3012845) on the redmedia line. Removed G-Rex100MCS flask from BSC, heated seal off the redcap from the red line near the terminal luer. Labeled G-Rex100MCS flaskwith QA provided in-process “Day 0” label. Attached sample “Day 0” labelbelow. Incubator parameters: 37.0±2.0° C.; CO₂ Percentage: 5.0±1.5% CO₂.

Placed the 50 mL conical tube” in incubator for ≥30 minutes of warming.

Day 0 Tumor Wash Media Preparation

Added Gentamicin to HBSS. In the BSC, added 5.0 mL Gentamicin (W3009832or W3012735) to 1×500 mL HBSS Media (W3013128) bottle. Recorded volumes.Added per bottle: HBSS (500.0 mL); Gentamicin sulfate, 50 mg/ml (5.0mL). Mixed reagents thoroughly. Filtered HBSS containing gentamicinprepared in Step 8.2.1 through a 1 L 0.22-micron filter unit (W1218810).Aseptically capped the filtered media and labeled with the followinginformation.

Day 0 Tumor Processing

Obtained tumor specimen and transferred into suite at 2-8° C.immediately for processing and recorded tumor information. Labeled three50 ml conical tubes: the first as “Forceps,” the second as “Scalpel,”and the third as “Fresh Tumor Wash Media”. Labeled 5×100 mm petri dishesas “Wash 1,” “Wash 2,” “Wash 3,” “Holding,” and “Unfavorable.” Labeledone 6 well plate as “Favorable Intermediate Fragments.”

Using an appropriately sized pipette, transferred 5.0 mL of “Tumor WashMedia” into each well of one 6-well plate for favorable intermediatetumor fragments (30.0 mL total). Using an appropriately sized pipette,transferred 50.0 mL of “Tumor Wash Media” prepared in Step 8.2.4 intoeach 100 mm petri dish for “Wash 1,” “Wash 2,” “Wash 3,” and “Holding”(200.0 mL total). Using an appropriately sized pipette, transfer 20.0 mLof “Tumor Wash Media” prepared in Step 8.2.4 into each 50 mL conical(60.0 mL total). Aseptically removed lids from two 6-well plates. Thelids were utilized for selected tumor pieces. Aseptically passed thetumor into the BSC. Recorded processing start time.

Tumor Wash 1: Using forcepts, removed the tumor from the specimen bottleand transferred to the “Wash 1”. Using forceps, gently washed tumor andrecord time. Transferred 20.0 mL (or available volume) of solution fromthe tumor specimen bottle into a 50 mL conical per sample plan. Labeledand stored bioburden sample collected at 2-8° C. until submitted fortesting.

Tumor Wash 2: Using a new set of forceps, removed the tumor from the“Wash 1” dish and transferred to the “Wash 2” dish. Using forceps,washed tumor specimen by gently agitating for ≥3 minutes and allowed itto sit. Recorded time.

Using a transfer pipette, placed 4 individual drops of Tumor Wash Mediafrom the conical into each of the 6 circles on the upturned lids of the6-well plates (2 lids). Placed an extra drop on two circles for a totalof 50 drops.

Tumor Wash 3: Using forceps, removed the tumor from the “Wash 2” dishand transferred to the “Wash 3” dish. Using forceps, washed tumorspecimen by gently agitating and allowed it to sit for ≥3 minutes.Recorded time.

Placed a ruler under 150 mm dish lid. Using forceps, asepticallytransferred tumor specimen to the 150 mm dissection dish lid. Arrangedall pieces of tumor specimen end to end and recorded the approximateoverall length and number of fragments. Assessed the tumor fornecrotic/fatty tissue. Assessed whether >30% of entire tumor areaobserved to be necrotic and/or fatty tissue; if yes, ensure tumor was ofappropriate size if so proceeded. Assessed whether <30% of entire tumorarea were observed to be necrotic or fatty tissue; if yes, proceeded.

Clean-Up Dissection. If tumor was large and >30% of tissue exterior wasobserved to be necrotic/fatty, performed “clean up dissection” byremoving necrotic/fatty tissue while preserving tumor inner structureusing a combination of scalpel and/or forceps. To maintain tumorinternal structure, used only vertical cutting pressure. Did not cut ina sawing motion with scalpel.

Using a combination of scalpel and/or forceps, cut the tumor specimeninto even, appropriately sized fragments (up to 6 intermediatefragments). To maintain tumor internal structure, use only verticalcutting pressure. Did not cut in a sawing motion with scalpel. Ensuredto keep non-dissected intermediate fragments completely submerged in“Tumor Wash Media”. Transferred each intermediate fragment to the“holding” dish

Manipulated one intermediate fragment at a time, dissected the tumorintermediate fragment in the dissection dish into pieces approximately3×3×3 mm in size, minimizing the amount of hemorrhagic, necrotic, and/orfatty tissues on each piece. To maintain tumor internal structure, usedonly vertical cutting pressure. Did not cut in a sawing motion withscalpel.

Selected up to eight (8) tumor pieces without hemorrhagic, necrotic,and/or fatty tissue. Used the ruler for reference. Continued dissectionuntil 8 favorable pieces have been obtained, or the entire intermediatefragment has been dissected. Transferred each selected piece to one ofthe drops of “Tumor Wash Media”.

After selecting up to eight (8) pieces from the intermediate fragment,placed remnants of intermediate fragment into a new single well of“Favorable Intermediate Fragments” 6-well plate.

If desirable tissue remains, selected additional Favorable Tumor Piecesfrom the “favorable intermediate fragments” 6-well plate to fill thedrops for a maximum of 50 pieces.

Recorded the total number of dissected pieces created.

Removed the “Tissue Pieces” 50 mL conical tube from the incubator.Ensured conical tube was warmed for ≥30 min. Passed “Tissue Pieces” 50mL conical into the BSC, ensuring not to compromise the sterility ofopen processing surfaces.

Using a transfer pipette, scalpel, forceps or combination, transferredthe selected 50 best tumor fragments from favorable dish lids to the“Tissue Pieces” 50 mL conical tube. If a tumor piece was dropped duringtransfer and desirable tissue remains, additional pieces from thefavorable tumor intermediate fragment wells were added. Recorded numbersof pieces.

Removed G-Rex100MCS containing media from incubator. Aseptically passedG-Rex100MCS flask into the BSC. When transferring the flask, did nothold from the lid or the bottom of the vessel. Transferred the vessel byhandling the sides. In the BSC, lifted G-Rex100MCS flask cap, ensuringthat sterility of internal tubing was maintained. Swirled conical tubewith tumor pieces to suspend and quickly poured the contents into theG-Rex100MCS flask.

Ensured that the tumor pieces were evenly distributed across themembrane of the flask. Gently tilted the flask back and forth ifnecessary to evenly distribute the tumor pieces. Recorded number oftumor fragments on bottom membrane of vessel and number of observed tobe floating in vessel. NOTE: If the number of fragments seeded were NOTequivalent to number of collected in Step 8.3.36H, contacted AreaManagement, and document in Section 10.0.

Incubated G-Rex100MCS at the following parameters: Incubated G-Rexflask: Temperature LED Display: 37.0±2.0° C.; CO₂ Percentage: 5.0±1.5%CO₂. Performed calculations to determine the proper time to removeG-Rex100MCS incubator on Day 11. Calculations: Time of incubation; lowerlimit=time of incubation+252 hours; upper limit=time of incubation+276hours.

Day 11—Media Preparation

Monitored Incubator. Incubator parameters: Temperature LED Display:37.0±2.0° C.; CO2 Percentage: 5.0±1.5% CO2. Warmed 3×1000 mL RPMI 1640Media (W3013112) bottles and 3×1000 mL AIM-V (W3009501) bottles in anincubator for ≥30 minutes. Recorded time. Media: RPMI 1640 and AIM-V.Placed an additional 1×1000 ml bottle of AIM-V Media (W3009501) at roomtemperature for further use.

Removed the RPMI 1640 Media when time was reached. Record end incubationtime in Step 8.4.4. Ensure media was warmed for ≥30 min. In the BSC,removed 100.0 mL from each of the three pre-warmed 1000 mL RPMI 1640Media bottles and placed into an appropriately sized container labeled“Waste”. In the BSC added the following reagents to each of the threeRPMI 1640 Media bottles and recorded volumes added to each bottle.GemCell Human serum, Heat Inactivated Type AB (100.0 mL), GlutaMax (10.0mL), Gentamicin sulfate, 50 mg/ml (1.0 mL), 2-mercaptoethanol (1.0 mL).

Caped bottles and swirled to ensure reagents were mixed thoroughly.Filtered each bottle of media through a separate 1 L 0.22-micron filterunit. Aseptically capped the filtered media and labeled each bottle withCM1 Day 11 Media. Thawed 3×1.1 mL aliquots of IL-2 (6×10⁶ IU/mL)(BR71424) until all ice had melted Recorded IL 2 lot # and Expiry.

Removed the three bottles of AIM-V Media from the incubator. Recordedend incubation time. Ensured media had been warmed for ≥30 minutes.Using a micropipette, added 3.0 mL of thawed IL-2 into one 1 L bottle ofpre-warmed AIM-V media. Rinse micropipette tip with media afterdispensing IL-2. Use a new sterile micropipette tip for each aliquot.Recorded the total volume added. Labeled bottle as “AIM-V ContainingIL-2”. Aseptically transferred a 10 L Labtainer Bag and a repeater pumptransfer set into the BSC. Closed all lines on a 10 L Labtainer bag.Attached the larger diameter tubing end of a repeater pump transfer setto the middle female port of the 10 L Labtainer Bag via luer lockconnection.

Staged the Baxa pump next to the BSC. Fed the transfer set tubingthrough the Baxa pump. Set the Baxa Pump to “High” and “9”. Removedsyringe from Pumpmatic Liquid-Dispensing System (PLDS) and discarded.Ensured to not compromise the sterility of the PLDS pipette.

Connected PLDS pipette to smaller diameter end of repeater pump fluidtransfer set via luer connection and placed pipette tip in AIM-V mediacontaining IL-2 bottle (prepared in Step 8.4.13) for aspiration. Openedall clamps between media bottle and 10 L Labtainer.

Using the PLDS, transfer pre-warmed AIM-V media containing IL-2 preparedin Step 8.4.13, as well as two additional AIM-V bottles into the 10 LLabtainer bag. Added the three bottles of filtered CM1 Day 11 Media fromStep 8.4.10. After addition of final bottle, cleared the line to thebag. NOTE: Stopped the pump between addition of each bottle of media.Removed PLDS from the transfer set and placed a red cap on the luer ofthe line in the BSC. Gently massaged the bag to mix. Labeled the mediabag with the following information. Expiration date was 24 hours fromthe preparation date.

Attached a 60 mL syringe to the available female port of the “CompleteCM2 Day 11 Media” bag. Removed 20.0 mL of media and place in a 50 mLconical tube. Placed a red cap on the female port of the “Complete CM2Day 11 Media” Bag. Labeled and stored Media Retain Sample at 2-8° C.until submitted for testing. Heat sealed off the red cap on the transferset line, close to red cap. Kept the transfer set on the bag.

In the BSC, added 4.5 mL of AIM-V Media that had been labelled with “ForCell Count Dilutions” and lot number to four 15 mL conical tubes.Labeled the tubes with the lot number and tube number (1-4). Labeled 4cryovials “Feeder” and vial number (1-4). Transferred any remaining2-mercaptoethanol, GlutaMax, and human serum from the BSC to 2-8° C.

Outside of the BSC, weld a 1 L Transfer Pack to the transfer setattached to the “Complete CM2 Day 11 Media” bag prepared. Labeledtransfer pack as “Feeder Cell CM2 Media” and lot number. Made a mark onthe tubing of the 1 L Transfer Pack tubing a few inches away from thebag. Placed the empty Transfer Pack onto the scale so that the tubingwas on the scale to the point of the mark. Tared the scale and left theempty Transfer Pack on the scale.

Set the Baxa pump to “Medium” and “4.” Pumped 500.0±5.0 mL of “CompleteCM2 Day 11” media prepared in Step 8.4.22 into Cell CM2 Media” transferpack. Measured by weight and recorded the volume of Complete CM2 mediaadded to the Transfer Pack.

Once filled, heated seal the line. Separated CM2 Day 11 media bag withtransfer set from feeder cell media transfer pack, kept weld toward 1 Ltransfer pack. Placed “Complete CM2 Day 11 Media” prepared in incubatoruntil use.

Day 11—TIL Harvest

Incubator parameters: Temperature LED Display: 37.0±2.0° C.; CO2Percentage: 5.0±1.5% CO2. Performed check to ensure incubationparameters are met before removing G-Rex100MCS from incubator. Lowerlimits the same as described above.

Recorded Time of Removal from incubator. Carefully removed G-Rex100MCSfrom incubator and ensured all clamps were closed except large filterline. Recorded processing start time.

Labeled a 300 mL Transfer pack as “TIL Suspension”. Sterile welded theTIL Suspension transfer (single line) of a Gravity Blood Filter. Placedthe 300 mL Transfer Pack on a scale and record dry weight. Labeled 1 LTransfer Pack as “Supernatant”.

Sterile welded the red media removal line from the G-Rex100MCS to the“Supernatant” transfer pack. Sterile welded the clear cell removal linefrom the G-Rex100MCS to one of the two spike lines on the top of theblood filter connected to the “TIL Suspension” transfer pack. PlacedG-Rex100MCS on the left side of the GatheRex and the “Supernatant” and“TIL Suspension” transfer packs to the right side.

Install the red media removal line from the G Rex100MCS to the top clamp(marked with a red line) and tubing guides on the GatheRex. Installedthe clear harvest line from the G-Rex100MCS to the bottom clamp (markedwith a blue line) and tubing guides on the GatheRex. Attached the gasline from the GatheRex to the sterile filter of the G-Rex100MCS flask.Before removing the supernatant from the G-Rex100MCS flask, ensured allclamps on the cell removal lines were closed. Transferred ˜900 mL ofculture supernatant from the G-Rex100MCS to the 1 L Transfer Pack.Visually inspected G-Rex100MCS flask to ensure flask is level and mediahas been reduced to the end of the aspirating dip tube.

After removal of the supernatant, closed all clamps to the red line.

Vigorously tapped flask and swirled media to release cells. Performed aninspection of the flask to ensure all cells have detached. NOTE:Contacted area management if cells did not detach. Tilted flask awayfrom collection tubing and allowed tumor pieces to settle along edge.Slowly tipped flask toward collection tubing so pieces remained on theopposite side of the flask. If the cell collection straw is not at thejunction of the wall and bottom membrane, rapping the flask while tiltedat a 450 angle is usually sufficient to properly position the straw.

Released all clamps leading to the TIL Suspension transfer pack. Usingthe GatheRex, transferred the cell suspension through the blood filterinto the 300 mL transfer pack. Maintained the tilted edge until allcells and media are collected. Inspected membrane for adherent cells.Rinsed the bottom of the G-Rex100MCS. Cover ˜¼ of gas exchange membranewith rinse media. Ensured all clamps are closed. Heat sealed (perProcess Note 5.12) the TIL suspension transfer pack as close to the weldas possible so that the overall tubing length remains approximately thesame. Heat sealed the “Supernatant” transfer pack. Maintained enoughline to weld. Recorded weight of TIL Suspension transfer pack andcalculated the volume of cell suspension.

Welded a 4″ plasma transfer set to “supernatant” transfer pack,retaining the luer connection on the 4″ plasma transfer set, andtransferred into the BSC. Welded a 4″ plasma transfer set to 300 mL “TILSuspension” transfer pack, retained the luer connection on the 4″ plasmatransfer set, and transferred into the BSC.

Drew up approximately 20.0 mL of supernatant from the 1 L “Supernatant”transfer pack and dispense into a sterile 50 mL conical tube labeled“Bac-T.” Removed a 1.0 mL sample from the 50 mL conical labeled BacTusing an appropriately sized syringe and inoculated the anaerobicbottle.

Labeled 4 cryovials with vial number (1-4). Using separate 3 mLsyringes, pulled 4×1.0 mL cell count samples from TIL SuspensionTransfer Pack using the luer connection, and placed in respectivecryovials. Placed a red cap (W3012845) on the line. Placed TIL TransferPack in incubator until needed. Perform cell counts and calculations.Perform initial cell counts undiluted. If no dilution needed, “Sample[μL]”=200, “Dilution [μL]”=0.

Record cell counts and TIL numbers. If Total Viable TIL Cells is <5×10⁶cells, proceeded to “Day 11 G-Rex Fill and Seed”. If Total Viable TILCells is >5×10⁶, proceed to “Calculation for flow cytometry”.

Calculation for Flow Cytometry.

If the Total Viable TIL Cell count was ≥4.0×10⁷, calculated the volumeto obtain 1.0×10⁷ cells for the flow cytometry sample. Total viablecells required for flow cytometry: 1.0×10⁷ cells. Volume of cellsrequired for flow cytometry: Viable cell concentration divided by1.0×10⁷ cells.

If Applicable: Recalculated Total Viable Cells and Volume flow.Calculated the remaining Total Viable Cells and remaining volume afterthe removal of cytometry sample below.

TIL Cryopreservation of Sample

If Applicable: Calculated Volume for Cryopreservation. Calculated thevolume of cells required to obtain 1×10⁷ cells for cryopreservation.

TABLE 24 Cryopreservation calculation Volume of Cells Total Viable TILrequired for required for Viable Cell cryopreservation cryopreservationConcentration C = A ÷ B A. 1 × 10⁷ cells B. cells/mL C. mL

If Applicable: Removed sample for Cryopreservation. Removed thecalculated volume from the TIL Suspension transfer pack. Placed inappropriately sized conical tube and label as “Cryopreservation Sample1×10⁷ cells,” dated, and lot number. Placed a red cap (W3012845) on theTIL Suspension transfer pack.

Centrifuged the “Cryopreservation Sample 1×10⁷ cells” according to thefollowing parameters: Speed: 350×g, Time: 10:00 minutes, Temperature:Ambient, Brake: Full (9); Acceleration: Full (9).

Added CS-10. In BSC, aseptically aspirate supernatant. Gently tap bottomof tube to resuspend cells in remaining fluid. Added CS-10. Slowly added0.5 mL of CS10. Recorded volume added. Cryopreservation Sample VialsFilled at ˜0.5 mL.

Day 11—Feeder Cells

Obtained 3 bags of feeder cells with at least two different lot numbersfrom LN2 freezer. Kept cells on dry ice until ready to thaw. Recordedfeeder cell information. Confirmed that at least two different lots offeeder cells were obtained. Placed the Feeder Cell bags into individualzip top bags, based on Lot number, and thawed 37.0±2.0° C. water bath orcytotherm for ˜3-5 minutes or until ice has just disappeared.

Feeder cell harness preparation. Welded 4S-4M60 to a CC2 Cell Connect(W3012820), replacing a single spike of the Cell Connect apparatus withthe 4-spike end of the 4S-4M60 manifold. Welded as needed.

Attached media transfer pack Weld the “Feeder Cell CM2 Media” transferpack to a CC2 luer. The bag will be attached to the side of the harnesswith the needless injection port. Transferred the assembly containingthe Complete CM2 Day 11 Media into the BSC.

Pool thawed feeder cells. Within the BSC, pulled 10 mL of air into a 100mL syringe. Used this to replace the 60 mL syringe on the CC2. Wipedeach port on the feeder cell bags with an alcohol pad prior to removingthe cover. Spike the three feeder bags using three of the spikes of theCC2. Maintained constant pressure while turning the spike in onedirection. Ensure to not puncture the side of the port. Opened thestopcock so that the line from the feeder cell bags is open and the lineto the needless injection port is closed. Drew up the contents of thefeeder cell bags into the syringe. All three bags drained at once. Oncefeeder cell bags had been drained, while maintaining pressure on thesyringe, clamped off the line to the feeder cell bags. Did not detachsyringe below. the syringe from the harness. Recorded the total volumeof feeder cells in the syringe.

Added feeder cells to transfer pack. Turned the stopcock so the line tothe feeder cell bag was closed and the line to the media Transfer Packwas open. Ensured the line to media transfer pack is unclamped.Dispensed the feeder cells from the syringe into the “Feeder Cell CM2Media” transfer pack. Clamped off the line to the transfer packcontaining the feeder cells and leave the syringe attached to theharness. Massaged bag to mix the pooled feeder cells in the transferpack. Labeled bag as “Feeder Cell Suspension”.

Calculated the total volume of feeder cell suspension. Removed cellcount samples. Using a separate 3 mL syringe for each sample, pulled4×1.0 mL cell count samples from Feeder Cell Suspension Transfer Packusing the needless injection port. Aliquoted each sample into labeledcryovials.

Performed cell counts and calculations utilizing NC-200 and Process Note5.14. Diluted cell count samples by adding 0.5 mL of cell suspensioninto 4.5 mL of AIM-V media labelled with the lot number and “For CellCount Dilutions”. This will give a 1:10 dilution.

Recorded Cell Count and Sample volumes. If Total Viable Cells are<5×10⁹, proceed. If Total Viable Cells are ≥5×10⁹, proceeded as abovefor higher cells counts. Obtained additional Feeder Cells as needed andadded to transfer pack as discussed above. Calculated the volume ofFeeder Cell Suspension that was required to obtain 5×10⁹ viable feedercells. Calculated the volume of excess feeder cells to remove. Rounddown to nearest whole number.

Removed excess feeder cells. In a new 100 mL syringe, pulled up 10 mL ofair and attached the syringe to the harness. Opened the line to the“Feeder Cell Suspension” transfer pack. Using the syringe drew up thevolume of feeder cells calculated in Step 8.6.71C plus an additional10.0 mL from the Transfer Pack into a 100 mL syringe. Closed the line tothe Feeder Cell Suspension transfer pack once the volume of feeder cellsis removed. Did not remove final syringe. Once a syringe has beenfilled, replaced it with a new syringe. Multiple syringes could be usedto remove total volume. With each new syringe, pulled in 10 mL of air.Recorded the total volume (including the additional 10 mL) of feedercells removed.

Added OKT3. In the BSC, using a 1.0 mL syringe and 16 G needle, drew up0.15 mL of OKT3. Aseptically removed the needle from the syringe andattach the syringe to the needless injection port. Injected the OKT3.Opened the stopcock to the “Feeder Cell Suspension” transfer pack andadded 10 mL of feeder cells removed in Step 8.6.73 to flush OKT3 throughthe line. Turned the syringe upside down and push air through to clearthe line to the Feeder Cell Suspension transfer pack. Left the remainingfeeder cell suspension in the syringe. Closed all clamps and remove theharness from the BSC. Heat sealed the Feeder Cell Suspension transferpack, leaving enough tubing to weld.

Day 11 G-Rex Fill and Seed

Set up G-Rex500MCS. Removed a G-Rex500MCS from packaging and inspectedthe flask for any cracks or kinks in the tubing. Ensured all luerconnections and closures were tight. Closed all clamps on theG-Rex500MCS lines except for the vent filter line. Using a marker drew aline at the 4.5 L gradation. Removed the “Complete CM2 Day 11 Media”,from the incubator.

Prepared to pump media. Welded the red line of the G-Rex500MCS to therepeater pump transfer set attached to the complete CM2 Day 11 Media.Hung the “Complete CM2 Day 11 Media” bag on an IV pole. Fed the pumptubing through the Baxa pump. Pumped media into G-Rex500MCS. Set theBaxa pump to “High” and “9”. Pumped 4.5 L of media into the G-Rex500MCS,filling to the line marked on the flask at the 4.5 L gradation. Heatsealed the red line of the G-Rex500MCS near the weld. Labeled the flaskwith the “Day 11” label. Welded the Feeder Cell: Suspension transferpack to the flask. Sterile welded the red line of the G-Rex500MCS to the“Feeder Cell Suspension” transfer pack.

Added Feeder Cells to G-Rex500MCS. Opened all clamps between Feeder CellSuspension and G-Rex500MCS and added Feeder Cell Suspension to flask bygravity feed. Heat sealed the red line near the weld. Welded the TILSuspension transfer pack to the flask. Sterile weld the red line of theG-Rex500MCS to the “TIL Suspension” transfer pack.

Added TIL to G-Rex500MCS. Opened all clamps between TIL Suspension andG-Rex500MCS and added TIL Suspension to flask by gravity feed. Heatsealed the red line near the weld to remove the TIL suspension bag.

Incubated G-Rex500MCS. Checked that all clamps on the G-Rex500MCS wereclosed except the large filter line and place in the incubator.Incubator parameters: Temperature LED Display: 37.0±2.0° C., CO2Percentage: 5.0±1.5% CO2.

Calculated incubation window. Performed calculations to determine theproper time to remove G-Rex500MCS from incubator on Day 16. Lower limit:Time of incubation+108 hours. Upper limit: Time of incubation+132 hours.

Day 11 Excess TIL Cryopreservation

Froze Excess TIL Vials. Recorded and verified the total number of vialsplaced into the Control Rate Freezer (CRF). Upon completion of freeze,transfer vials from CRF to the appropriate storage container.

Day 16 Media Preparation

Pre-warmed AIM-V Media. Removed three CTS AIM V 10 L media bags from2-8° C. at least 12 hours prior to use and place at room temperatureprotected from light. Labeled each bag. Record warming start time anddate. Ensured all bags have been warmed for a duration between 12 and 24hours.

Attached the larger diameter end of a fluid pump transfer set to one ofthe female ports of a 10 L Labtainer bag using the Luer connectors.Setup 10 L Labtainer for Supernatant Label as “Supernatant”. Setup 10 LLabtainer for Supernatant. Ensure all clamps were closed prior toremoving from the BSC.

Thawed 5×1.1 mL aliquots of IL-2 (6×10⁶ IU/mL) (BR71424) per bag of CTSAIM V media until all ice had melted. Aliquoted 100.0 mL of Glutamaxinto an appropriately sized receiver. Recorded the volume added to eachreceiver and labeled each receiver as “GlutaMax.”

Added IL-2 to GlutaMax. Using a micropipette, added 5.0 mL of IL-2 toeach GlutaMax receiver. Ensured to rinse the tip per process note 5.18and used a new pipette tip for each mL added. Recorded volume added toeach Glutamax receiver and labeled each receiver as “GlutaMax+IL-2” andreceiver number.

Prepared CTS AIM V media bag for formulation. Ensured CTS AIM V 10 Lmedia bag (W3012717) was warmed at room temperature and protected fromlight for 12-24 hours prior to use. Recorded end incubation time. In theBSC, closed clamp on a 4″ plasma transfer set, then connected to the bagusing the spike ports. Maintained constant pressure while turning thespike in one direction. Ensured to not puncture the side of the port.Connected the larger diameter end of a repeater pump fluid transfer setto the 4″ plasma transfer set via luer.

Stage Baxa pump next to BSC. Removed pump tubing section of repeaterpump fluid transfer set from BSC and installed in repeater pump.

Prepared to formulate media. In BSC, removed syringe from PumpmaticLiquid-Dispensing System (PLDS) and discarded. Ensured to not compromisethe sterility of the PLDS pipette. Connected PLDS pipette to smallerdiameter end of repeater pump fluid transfer set via luer connection andplaced pipette tip in “GlutaMax+IL-2” prepared above for aspiration.Open all clamps between receiver and 10 L bag.

Pumped GlutaMax+IL-2 into bag. Set the pump speed to “Medium” and “3”and pump all “GlutaMax+IL-2” into 10 L CTS AIM V media bag. Once nosolution remains, clear line and stop pump. Recorded the volume ofGlutaMax containing IL-2 added to each Aim V bag below.

Removed PLDS. Ensured all clamps were closed, and removed the PLDSpipette from the repeater pump fluid transfer set. Removed repeater pumpfluid transfer set and red cap the 4″ plasma transfer set.

Labeled each bag of “Complete CM4 Day 16 media” prepared.

Removed Media Retain per Sample Plan. Using a 30 mL syringe, removed20.0 mL of “Complete CM4 Day 16 media” by attaching syringe to the 4″plasma transfer set and dispensed sample into a 50 mL conical tube.Ensure 4″ plasma transfer set was either clamped or red capped afterremoval of syringe.

Attached new repeater pump fluid transfer set. Attached the largerdiameter end of a new fluid pump transfer set onto the 4″ plasmatransfer set that was connected to the “Complete CM4 Day 16 media” bag.Labeled with sample plan inventory label and stored media retain sampleat 2-8° C. until submitted for testing.

Monitored Incubator. If applicable, monitor for additional bagsprepared. Incubator parameters: Temperature LED Display: 37.0±2.0° C.,CO2 Percentage: 5.0±1.5% CO2.

Warmed Complete CM4 Day 16 Media. Warmed the first bag of Complete CM4Day 16 Media in incubator for ≥30 minutes until ready for use. Ifapplicable, warmed additional bags.

Prepared Dilutions. In the BSC, added 4.5 mL of AIM-V Media that hadbeen labelled with “For Cell Count Dilutions” to each 4×15 mL conicaltube. Labeled the conical tubes. Labeled 4 cryovials.

Day 16 REP Spilt

Monitored Incubator. Incubator parameters: Temperature LED Display:37.0±2.0° C., CO2 Percentage: 5.0±1.5% CO2

Removed G-Rex500MCS from Incubator. Performed check below to ensureincubation parameters are met before removing G-Rex500MCS fromincubator: upper limit, lower limit, time of removal. RemovedG-Rex500MCS from the incubator.

Heat sealed a 1 L transfer pack (W3006645), leaving ˜12″ of line.Labeled 1 L transfer pack as TIL Suspension. Place 1 L transfer pack,including the entire line, on a scale and record dry weight.

GatheRex Setup. Sterile welded the red media removal line from theG-Rex500MCS to the repeater pump transfer set on the 10 L labtainer bag“Supernatant” prepared above. Sterile welded the clear cell removal linefrom the G-Rex500MCS to the TIL Suspension transfer pack prepared above.Placed G-Rex500MCS flask on the left side of the GatheRex. Placed thesupernatant labtainer bag and TIL suspension transfer pack to the rightside. Installed the red media removal line from the G-Rex500MCS to thetop clamp (marked with a red line) and tubing guides on the GatheRex.Installed the clear harvest line from the G-Rex500MCS to the bottomclamp (marked with a blue line) and tubing guides on the GatheRex.Attached the gas line from the GatheRex to the sterile filter of theG-Rex500 MCS. NOTE: Before removing the supernatant from theG-Rex500MCS, ensured all clamps on the cell removal lines were closed.

Volume Reduction of G-Rex500MCS. Transferred ˜4.5 L of culturesupernatant from the G-Rex500MCS to the 10 L Labtainer per SOP-01777.Visually inspect G-Rex500MCS to ensure flask as level and media had beenreduced to the end of the aspirating dip tube.

Prepared flask for TIL Harvest. After removal of the supernatant, closedall clamps to the red line.

Initiation of TIL Harvest. Recorded the start time of the TIL harvest.Vigorously tap flask and swirl media to release cells. Performed aninspection of the flask to ensure all cells have detached. Tilted theflask to ensure hose is at the edge of the flask. If the cell collectionstraw is not at the junction of the wall and bottom membrane, rappingthe flask while tilted at a 450 angle is usually sufficient to properlyposition the straw.

TIL Harvest. Released all clamps leading to the TIL suspension transferpack. Using the GatheRex transferred the cell suspension into the TILSuspension transfer pack. NOTE: Be sure to maintain the tilted edgeuntil all cells and media are collected. Inspected membrane for adherentcells.

Rinsed flask membrane. Rinsed the bottom of the G-Rex500MCS. Cover ˜¼ ofgas exchange membrane with rinse media. Closed clamps on G-Rex500MCS.Ensured all clamps were closed on the G-Rex500MCS.

Heat sealed. Heat sealed the Transfer Pack containing the TIL as closeto the weld as possible so that the overall tubing length remainedapproximately the same. Heat sealed the 10 L Labtainer containing thesupernatant and passed into the BSC for sample collection.

Recorded weight of Transfer Pack with cell suspension and calculate thevolume suspension. Prepared transfer pack for sample removal. Welded a4″ Plasma Transfer Set, to the TIL Suspension transfer pack from above,leaving the female luer end attached as close to the bag as possible.

Removed testing samples from cell supernatant. In the BSC, remove 10.0mL of supernatant from 10 L labtainer using female luer port andappropriately sized syringe. Placed into a 15 mL conical tube and labelas “BacT” and Retain the tube for BacT sample. Using a separate syringe,removed 10.0 mL of supernatant and placed into a 15 mL conical tube.Retained the tube for mycoplasma sample for testing. Labeled tube as“Mycoplasma diluent”. Closed supernatant bag. Placed a red cap on theluer port to close the bag, and pass out of BSC.

Removed Cell Count Samples. In the BSC, using separate 3 mL syringes foreach sample, removed 4×1.0 mL cell count samples from “TIL Suspension”transfer pack using the luer connection. Placed samples in cryovialsprepared above.

Removed Mycoplasma Samples. Using a 3 mL syringe, removed 1.0 mL fromTIL Suspension transfer pack and place into 15 mL conical labeled“Mycoplasma diluent” prepared above. Labeled and stored Mycoplasmasample at 2-8° C. until submitted for testing.

Prepared Transfer Pack for Seeding. In the BSC, attached the largediameter tubing end of a Repeater Pump Fluid Transfer Set to the Lueradapter on the transfer pack containing the TIL. Clamped the line closeto the transfer pack using a hemostat. Placed a red cap onto the end ofthe transfer set.

Placed TIL in Incubator. Removed cell suspension from the BSC and placein incubator until needed. Recorded time.

Performed Cell Counts. Performed cell counts and calculations utilizingNC-200. Diluted cell count samples initially by adding 0.5 mL of cellsuspension into 4.5 mL of AIM-V media prepared above. This gave a 1:10dilution.

Calculated flasks for subculture. Calculated the total number of flasksto seed. NOTE: Rounded the number of G-Rex500MCS flasks to see up to theneared whole number.

TABLE 25 Flask calculation Number Target of G-Rex500MCS Total ViableCell Count Cells Required per Flask Flasks to Seed A B C = A ÷ B cells1.0 × 10⁹ cells/flask flasks

The maximum number of G-Rex500MCS flasks to seed was five. If thecalculated number of flasks to seed exceeded five, only five were seededUSING THE ENTIRE VOLUME OF CELL SUSPENSION AVAILABLE.

Determined number of additional media bags needed. Calculated the numberof media bags required in addition to the bag prepared above. Round thenumber of media bags required up to the next whole number.

TABLE 26 Media bag calculation Number Number of Number of Bags Number ofof G-Rex500MCS Media Bag Prepared in Additional Bags Flasks to SeedRequired above to Prepare A B = A ÷ 2* C D = B − C 1

Prepared additional media as needed. Prepared one 10 L bag of “CM4 Day16 Media” for every two G-Rex-500M flask needed calculated in Step8.10.59D. Proceeded to Step 8.10.62 and seeded the first GREX-500Mflask(s) while additional media is prepared and warmed.

Prepared additional media bags as needed. Prepared and warmed thecalculated number of additional media bags determined above.

Filled G-Rex500MCS. Opened a G-Rex500MCS on the benchtop and inspectedfor cracks in the vessel or kinks in the tubing. Ensured all luerconnections and closures were tight. Made a mark at the 4500 mL line onthe outside of the flask with a marker. Closed all clamps on theG-Rex500MCS except the large filter line. Sterile welded the red medialine of a G-Rex500MCS to the fluid transfer set on the media bagprepared above.

Prepared to pump media. Hung “CM4 Day 16 Media” on an IV pole. Fed thepump tubing through the Baxa pump.

Pumped media into G-Rex500MCS. Set the Baxa pump on “High” and “9” andpump 4500 mL of media into the flask. Pumped 4.5 L of “CM4 Day 16 Media”into the G-Rex500MCS, filling to the line marked on the flask as above.Once 4.5 L of media had been transferred, stopped the pump.

Heat Sealed. Heat sealed the red media line of G-Rex500MCS, near theweld created, removing the media bag.

Repeated Fill. Repeat filling and sealing steps for each flaskcalculated in above as media is warmed and prepared for use. Multipleflasks may be filled at the same time using gravity fill or multiplepumps. Fill only two flasks per bag of media.

Recorded and labelled flask(s) filled. Labeled each flask alphabeticallyand with “Day 16” labels.

As needed incubated flask. Held flask in incubator while waiting to seedwith TIL. Recorded the total number of flasks filled.

Calculated volume of cell suspension to add. Calculated the targetvolume of TIL suspension to add to the new G-Rex500MCS flasks.

TABLE 27 Cell suspension volume Target Volume of cell Total Volume ofTIL suspension to transfer suspension to each flask A Number of flask(s)filled C = A ÷ B mL mL

If number of flasks exceeds five only five will be seeded, USING THEENTIRE VOLUME OF CELL SUSPENSION.

Prepared Flasks for Seeding. Removed G-Rex500MCS from Step 8.10.70 fromthe incubator.

Prepared for pumping. Closed all clamps on G-Rex500MCS except largefilter line. Fed the pump tubing through the Baxa pump.

Removed TIL from incubator. Removed “TIL Suspension” transfer pack fromthe incubator and record incubation end time.

Prepared cell suspension for seeding. Sterile welded “TIL Suspension”transfer pack from above to pump inlet line.

Placed TIL suspension bag on a scale. Primed the line from the TILsuspension bag to the weld using the Baxa pump set to “Low” and “2”.Tared the scale.

Seeded flask with TIL Suspension. Set Baxa pump to “Medium” and “5”.Pump the volume of TIL suspension calculated above into flask. Recordthe volume of TIL Suspension added to each flask.

Heat sealed. Heat sealed the “TIL Suspension” transfer pack, leavingenough tubing to weld on the next flask.

Filled remaining flasks. Between each flask seeded, ensured to mix “TILSuspension” transfer pack and repeat filling and sealing steps to seedall remaining flaks.

Monitored Incubator. If flasks must be split among two incubators,ensure to monitor both. Incubator parameters: Temperature LED Display:37.0±2.0° C., CO₂ Percentage: 5.0±1.5% CO₂. Recorded the time each flaskis placed in the incubator.

Calculated incubation window. Performed calculations below to determinethe time range to remove G-Rex500MCS from incubator on Day 22. Lowerlimit: time+132 hours; upper limit: time+156 hours.

Day 22 Wash Buffer Preparation

Prepared 10 L Labtainer Bag. In BSC, attach a 4″ plasma transfer set toa 10 L Labtainer Bag via luer connection. Prepared 10 L Labtainer BagLabel as “Supernatant”, lot number, and initial/date. Closed all clampsbefore transferring out of the BSC. NOTE: Prepared one 10 L LabtainerBag for every two G-Rex500MCS flasks to be harvested.

Welded fluid transfer set. Outside the BSC, closed all clamps on4S-4M60. Welded repeater fluid transfer set to one of the male luer endsof 4S-4M60.

Passed Plasmalyte-A and Human Albumin 25% into the BSC. Passed the4S-4M60 and repeater fluid transfer set assembly into the BSC.

TABLE 28 Components Component Description Amount Needed Plasmalyte-A3000.0 mL Human Albumin 25% 120.0 mL 4S-4M60 with Repeater 1 ApparatusFluid Transfer Set Step 8.11.7

TABLE 29 Plasmalyte-A Latex: Not Made with Natural Rubber LatexContainer Type: VIAFLEX PVC: Contains PVC DEHP: Contains DEHP Volume:500 ML Total Calories: 21 Kcal/L Sodium: 140 mEq/L Potassium: 5 mEq/LMagnesium: 3 mEq/L Acetate: 27 mEq/L Chloride: 98 mEq/L Gluconate: 23mEq/L Osmolarity (mOsmol/L): 294 Specific Gravity: 1.01 pH: 7.4 FillRange Volume (mL): 530-565 Shelf Life from manufacture: 15 monthsContains Preservative: No Storage Recommendations: Store at roomtemperature (25° C./77° F.); brief exposure up to 40° C./104° F. doesnot adversely affect the product. Packaging: Single Pack Rx Only: Yes**As commercially available fromhttp://ecatalog.baxter.com/ecatalog/loadproduct.html?cid=20016&lid=10001&hid=20001&pid=821874.

Pumped Plasmalyte into 3000 mL bag. Spiked three bags of Plasmalyte-A tothe 4S-4M60 Connector set. NOTE: Wipe the port cover with an alcoholswab (W3009488) prior to removing. NOTE: Maintain constant pressurewhile turning the spike in one direction. Ensure to not puncture theside of the port. Connected an Origen 3000 mL collection bag via luerconnection to the larger diameter end of the repeater pump transfer set.Closed clamps on the unused lines of the 3000 mL Origen Bag. Staged theBaxa pump next to the BSC. Fed the transfer set tubing through the Baxapump situated outside of the BSC. Set pump to “High” and “9”. Opened allclamps from the Plasmalyte-A to the 3000 mL Origen Bag. Pumped all ofthe Plasmalyte-A into the 3000 mL Origen bag. Once all the Plasmalyte-Ahad been transferred, stopped the pump. If necessary, removed air from3000 mL Origen bag by reversing the pump and manipulating the positionof the bag. Closed all clamps.

Remove the 3000 mL bag from the repeater pump fluid transfer set vialuer connection and placed a red cap (W3012845) on the line to the bag.

Added Human Albumin 25% to 3000 mL Bag. Opened vented mini spike.Without compromising sterility of spike, ensured blue cap is securelyfastened. Spiked the septum of a Human Albumin 25% bottle with thevented mini spike. NOTE: Ensured to not compromise the sterility of thespike. Repeated two times for a total of three (3) spiked Human Albumin25% bottles. Removed the blue cap from one vented mini spike and attacha 60 mL syringe to the Human Serum Albumin 25% bottle. Draw up 60 mL ofHuman Serum Albumin 25%. It may be necessary to use more than one bottleof Human Serum Albumin 25%. If necessary, disconnect the syringe fromthe vented mini spike and connect it to the next vented mini spike in aHuman Serum Albumin 25% bottle. Once 60 mL has been obtained, remove thesyringe from the vented mini spike. Attach syringe to needlelessinjection port on 3000 mL Origen bag filled with Plasmalyte-A. Dispensedall of the Human Albumin 25%. Repeated to obtain a final volume of 120.0mL of Human Albumin 25%. Gently mixed the bag after all of the HumanAlbumin 25% had been added. Labeled as “LOVOWash Buffer” and assign a 24hour expiry.

Prepared IL-2 Diluent. Using a 10 mL syringe, removed 5.0 mL of LOVOWash Buffer using the needleless injection port on the LOVO Wash Bufferbag. Dispensed LOVO wash buffer into a 50 mL conical tube and label as“IL-2 Diluent”.

CRF Blank Bag LOVO Wash Buffer Aliquotted. Using a 100 mL syringe, drewup 70.0 mL of LOVO Wash Buffer from the needleless injection port. NOTE:Wiped the needless injection port with an alcohol pad before each use.Placed a red cap on the syringe and label as “blank cryo bag” and lotnumber. NOTE: Held the syringe at room temp until needed in Step 8.14.3

Completed Wash Buffer Prep. Closed all clamps on the LOVO Wash Bufferbag.

Thawed IL-2. Thawed one 1.1 mL of IL-2 (6×10⁶ IU/mL), until all ice hasmelted. Record IL-2 Lot number and Expiry. NOTE: Ensured IL-2 label isattached.

IL-2 Preparation. Added 50 L IL-2 stock (6×10⁶ IU/mL) to the 50 mLconical tube labeled “IL-2 Diluent.”

IL-2 Preparation. Relabeled conical as “IL-2 6×104”, the date, lotnumber, and 24 hour expiry. Cap and store at 2-8° C.

Cryopreservation Prep. Placed 5 cryo-cassettes at 2-8° C. toprecondition them for final product cryopreservation.

Prepared Cell Count Dilutions. In the BSC, added 4.5 mL of AIM-V Mediathat has been labelled with lot number and “For Cell Count Dilutions” to4 separate 15 mL conical tubes and labeled the tubes.

Prepared Cell Counts. Labeled 4 cryovials with vial number (1-4).

Day 22 TIL Harvest

Monitored the incubator. Incubator Parameters Temperature LED display:37±2.0° C., CO₂ Percentage: 5%+1.5%.

Removed G-Rex500MCS Flasks from Incubator. Check flasks and confirmincubation parameters were met before removing G-Rex500MCS fromincubator (incubation time).

Prepared TIL collection bag Labeled a 3000 mL collection bag as “TILSuspension”, lot number, and initial/date.

Sealed off extra connections. Heat sealed off two leur connections onthe collection bag near the end of each connection.

GatheRex Setup. Sterile welded (per Process Note 5.11) the red mediaremoval line from the G-Rex500MCS to the 10 L labtainer bag preparedabove. NOTE: Referenced Process Note 5.16 for use of multiple GatheRexdevices. Sterile welded (per Process Note 5.11) the clear cell removalline from the G-Rex500MCS to the TIL Suspension collection bag preparedabove. Placed the G-Rex500MCS flask on the left side of the GatheRex.Placed the supernatant Labtainer bag and pooled TIL suspensioncollection bag to the right side. Installed the red media removal linefrom the G-Rex500MCS to the top clamp (marked with a red line) andtubing guides on the GatheRex. Installed the clear harvest line from theG-Rex500MCS to the bottom clamp (marked with a blue line) and tubingguides on the GatheRex. Attached the gas line from the GatheRex to thesterile filter of the G-Rex500MCS. Before removing the supernatant fromthe G-Rex500MCS, ensured all clamps on the cell removal lines wereclosed.

Volume Reduction. Transferred ˜4.5 L of supernatant from the G-Rex500MCSto the Supernatant bag. Visually inspected G-Rex500MCS to ensure flaskis level and media had been reduced to the end of the aspirating diptube. Repeat step if needed.

Prepared flask for TIL Harvest. After removal of the supernatant, closedall clamps to the red line.

Initiated collection of TIL. Recorded the start time of the TIL harvest.Vigorously tap flask and swirl media to release cells. Performed aninspection of the flask to ensure all cells have detached. Placed “TILSuspension” 3000 mL collection bag on dry wipes on a flat surface.Tilted the flask to ensure hose is at the edge of the flask. NOTE: Ifthe cell collection hose was not at the junction of the wall and bottommembrane, rapping the flask while tilted at a 450 angle is usuallysufficient to properly position the hose.

TIL Harvest. Released all clamps leading to the TIL suspensioncollection bag. Using the GatheRex, transferred the TIL suspension intothe 3000 mL collection bag. NOTE: Maintained the tilted edge until allcells and media were collected. Inspect membrane for adherent cells.

Rinsed flask membrane. Rinsed the bottom of the G-Rex500MCS. Covered ˜¼of gas exchange membrane with rinse media.

Close dclamps on G-Rex500MCS. Ensure all clamps are closed.

Heat sealed. Heat seal the collection bag containing the TIL as close tothe weld as possible so that the overall tubing length remainedapproximately the same. Heat sealed the Supernatant bag.

Completed harvest of remaining G-Rex 500 MCS flasks. Repeat steps above,pooling all TIL into the same collection bag. It was necessary toreplace the 101 supernatant bag after every 2nd flask.

Prepared LOVO source bag. Obtained a new 3000 mL collection bag. Labeledas “LOVO Source Bag”, lot number, and Initial/Date. Heat sealed thetubing on the “LOVO Source bag”, removing the female luers, leavingenough line to weld.

Weighed LOVO Source Bag. Placed an appropriately sized plastic bin onthe scale and tare. Place the LOVO Source Bag, including ports andlines, in the bin and record the dry weight.

Transferred cell suspension into LOVO source bag. Closed all clamps of a170 m gravity blood filter.

Transferred cell suspension into LOVO source bag. Sterile welded thelong terminal end of the gravity blood filter to the LOVO source bag.Sterile welded one of the two source lines of the filter to “pooled TILsuspension” collection bag. Once weld was complete, heat sealed theunused line on the filter to remove it. Opened all necessary clamps andelevate the TIL suspension by hanging the collection bag on an IV poleto initiate gravity-flow transfer of TIL through the blood filter andinto the LOVO source bag. Gently rotated or knead the TIL Suspension bagwhile draining in order to keep the TIL in even suspension.

Closed all clamps. Once all TIL were transferred to the LOVO source bag,closed all clamps.

Heat Sealed. Heat sealed (per Process Note 5.12) as close to weld aspossible to remove gravity blood filter.

Removed Cell Counts Samples. In the BSC, using separate 3 mL syringesfor each sample, removed 4×1.0 mL cell count samples from the LOVOsource bag using the needless injection port. Placed samples in thecryovials prepared in Step 8.11.36.

Performed Cell Counts. Performed cell counts and calculations utilizingNC-200. Diluted cell count samples initially by adding 0.5 mL of cellsuspension into 4.5 mL of AIM-V media prepared above. This gave a 1:10dilution.

Recorded Cell Count and Sample Volumes. Calculated Total Viable TILCells. If Total Viable cells ≥1.5×10⁹, proceeded. Calculate TotalNucleated Cells.

Prepared Mycoplasma Diluent. In the BSC, removed 10.0 mL from onesupernatant bag via luer sample port and placed in a 15 mL conical.Label 15 mL conical “Mycoplasma Diluent”.

LOVO

Turned on the LOVO and started the “TIL G-Rex Harvest” protocol andfollowed screen prompts. Buffer type was PlasmaLyte. Followed the LOVOtouch screen prompts.

Determined the final product target volume. Using the total nucleatedcells (TNC) value and the chart below, determined the final producttarget volume and recorded (mL).

TABLE 30 Calculate final product volume Final Product (Retentate) Volumeto Cell Range Target (mL) 0 < Total (Viable + Dead) 165 Cells ≤ 7.1 ×10¹⁰ 7.1 × 10¹⁰ < Total (Viable + Dead) 215 Cells ≤ 1.1 × 10¹¹ 1.1 ×10¹¹ < Total (Viable + Dead) 265 Cells ≤ 1.5 × 10¹¹

Followed the LOVO touch screen prompts.

Loaded disposable kit. Prior to loading the disposable kit, wipepressure sensor port with an alcohol wipe followed by a lint-free wipe.Load the disposable kit. Follow screen directions on loading thedisposable kit.

Removed filtrate bag. When the standard LOVO disposable kit had beenloaded, touched the Next button. The Container Information and LocationScreen displayed. Removed filtrate bag from scale

Ensured Filtrate Container was New and Off-Scale

Entered Filtrate capacity. Sterile welded a LOVO Ancillary Bag onto themale luer line of the existing Filtrate Bag. Ensured all clamps are openand fluid path is clear. Touch the Filtrate Container Capacity entryfield. A numeric keypad displays. Enter the total new Filtrate capacity(5,000 mL). Touch the button to accept the entry. NOTE: EstimatedFiltrate Volume should not exceed 5000 mL.

Placed Filtrate container on benchtop. NOTE: If tubing was removed fromthe F clamp during welding, placed the tubing back into the clamp.Placed the new Filtrate container on the benchtop. DID NOT hang theFiltrate bag on weigh scale #3. Weigh scale #3 will be empty during theprocedure.

Followed the LOVO touch screen prompts after changes to the filtratecontainer.

Ensured kit was loaded properly. The Disposable Kit Dry Checks overlaydisplays. Checked that the kit was loaded properly and all clamps wereopen. Checked all tubing for kinks or other obstructions and correct ifpossible. Ensured kit was properly installed and check all Robert'sclamps. Pressed the Yes button. All LOVO mechanical clamps closedautomatically and the Checking Disposable Kit Installation screendisplays. The LOVO went through a series of pressurizing steps to checkthe kit.

Kit Check Results. If the Kit check passed, proceeded to the next step.*If No, a second Kit Check could be performed after checks have beencomplete. *If No, Checked all tubing for kinks or other obstructions andcorrect *If No, Ensured kit was properly installed and check allRobert's clamps. If the 2nd kit check failed: Contact area managementand prepare to installation of new kit in Section 10.0. Repeat Step8.13.23-Step 8.13.30 needed.

Attached PlasmaLyte. The Connect Solutions screen displayed. The washvalue would always be 3000 mL. Entered this value on screen.

Sterile welded the 3000 mL bag of PlasmaLyte to the tubing passingthrough Clamp 1. Hung the PlasmaLyte bag on an IV pole placing bothcorner bag loops on the hook.

Verified that the PlasmaLyte was attached. Opened any plastic clamps.Verified that the Solution Volume entry was 3000 mL. Touched the “Next”button. The Disposable Kit Prime overlay displayed. Verified that thePlasmaLyte was attached and any welds and plastic clamps on the tubingleading to the PlasmaLyte bag were open, then touched the Yes button

Observed that the PlasmaLyte is moving. Disposable kit prime starts andthe Priming Disposable Kit Screen displays. Visually observed thatPlasmaLyte moving through the tubing connected to the bag of PlasmaLyte.If no fluid was moving, pressed the Pause Button on the screen anddetermined if a clamp or weld was still closed. Once the problem hadbeen solved, pressed the Resume button on the screen to resume theDisposable Kit Prime. Followed the LOVO touch screen prompts.

Attached Source container to tubing. Sterile weld the LOVO Source Bagprepared in Step 8.12.31 to the tubing passing through Clamp S perProcess Note 5.11. It could be necessary to remove the tubing from theclamp. Note: Made sure to replace source tubing into the S clamp ifremoved.

Hung Source container. Hung the Source container on the IV pole placingboth corner bag loops on the hook. DID NOT hang the Source on weighscale #1. Opened all clamps to the source bag.

Verified Source container was attached. Touched the Next button. TheSource Prime overlay displayed. Verified that the Source was attached tothe disposable kit, and that any welds and plastic clamps on the tubingleading to the Source were open. Touched the Yes button.

Confirm PlasmaLyte was moving. Source prime started and the PrimingSource Screen displayed. Visually observed that PlasmaLyte is movingthrough the tubing attached to the Source bag. If no fluid is moving,press the Pause Button on the screen and determine if a clamp or weld isstill closed. Once the problem was solved, pressed the Resume button onthe screen to resume the Source Prime.

Started Procedure Screen. When the Source prime finishes successfully,the Start Procedure Screen displays. Pressed Start, the “Pre-Wash Cycle1” pause screen appears immediately after pressing start.

Inverted In Process Bag. Removed the In Process Bag from weigh scale #2(can also remove tubing from the In Process top port tubing guide) andmanually invert it to allow the wash buffer added during the disposablekit prime step to coat all interior surfaces of the bag. Re-hang the InProcess Bag on weigh scale #2 (label on the bag was facing to the left).Replace the top port tubing in the tubing guide, if it was removed.

Inverted Source bag. Before pressing the Start button, mixed the Sourcebag without removing it from the IV pole by massaging the bag cornersand gently agitating the cells to create a homogeneous cell suspension.Pressed the Resume button. The LOVO started processing fluid from theSource bag and the Wash Cycle 1 Screen displays.

Source Rinse Pause. The Rinse Source Pause screen displayed once thesource container was drained and the LOVO had added wash buffer to theSource bag. Without removing the Source bag from IV pole, massaged thecorners and mixed well. Pressed Resume.

Mixed In Process Bag Pause. To prepare cells for another pass throughthe spinner, the In Process Bag was diluted with wash buffer. Afteradding the wash buffer to the In Process Bag, the LOVO pausesautomatically and displays the “Mix In Process Bag” Pause Screen.Without removing the bag from the weigh scale, mixed the product well bygently squeezing the bag. Press Resume.

Massaged In Process Corners Pause. When the In Process Bag was empty,wash buffer was added to the bottom port of the In Process Bag to rinsethe bag. After adding the rinse fluid, the LOVO paused automatically anddisplayed the “Massage IP corners” Pause Screen. When the “Massage IPcorners” Pause Screen displayed, DO NOT remove the bag from weigh scale#2. With the In Process Bag still hanging on weigh scale #2, massage thecorners of the bag to bring any residual cells into suspension. Ensuredthe bag was not swinging on the weigh scale and pressed the Resumebutton.

Waited for Remove Products Screen. At the end of the LOVO procedure, theRemove Products Screen displayed. When this Screen displays, all bags onthe LOVO kit could be manipulated. Note: Did not touch any bags untilthe Remove Products displayed.

Removed retentate bag. Placed a hemostat on the tubing very close to theport on the Retentate bag to keep the cell suspension from settling intothe tubing. Heat sealed (per Process Note 5.12) below the hemostat,making sure to maintain enough line to weld in Step 8.13.48. Removed theretentate bag.

Prepared retentate bag for formulation. Welded the female luer lock endof a 4″ Plasma Transfer Set to the retentate bag. Transferred theretentate bag.

Removed Products. Followed the instructions on the Remove ProductsScreen. Closed all clamps on the LOVO kit to prevent fluid movement.

Removed Products. Touched the Next button. All LOVO mechanical clampsopened and the Remove Kit Screen displayed.

Recorded Data. Followed the instructions on the Remove Kit screen.Touched the “Next” button. All LOVO mechanical clamps close and theResults Summary Screen displays. Recorded the data from the resultssummary screen. Closed all pumps and filter support. Removed the kitwhen prompted to do so by the LOVO. All Times recorded were recordeddirectly from the LOVO.

Final Formulation and Fill

Target volume/bag calculation. From Table 31 below, selected the numberof CS750 bags to be filled, target fill volume per bag, volume removedfor retain per bag, and final target volume per bag that corresponded tothe Volume of LOVO Retentate from above.

TABLE 31 Target volume/bag calculation Final Volume Volume PredictedTarget Volume Final of of CS10 Volume of Number Fill removed Target LOVOto add to formulated of bags Volume for retain Volume product productproduct to be per bag per bag per bag 165 mL 165 mL 330 mL 3 107 mL 7 mL100 mL 215 mL 215 mL 430 mL 4 105 mL 5 mL 100 mL 265 mL 265 mL 530 mL 4130 mL 5 mL 125 mL

Prepared CRF Blank. Calculated volume of CS-10 and LOVO wash buffer toformulate blank bag.

TABLE 32 Calculated volumes. Final Target Blank LOVO Wash Blank CS-10Volume per Bag Buffer Volume Volume (mL) A B = A/2 C = B mL mL mL

Prepared CRF Blank. Outside of the BSC, using the syringe of LOVO WashBuffer prepared in above, added volume calculated to an empty CS750 bagvia luer connection. Note: Blank CS750 bag formulation does not need tobe done aseptically. Using an appropriately sized syringe, added thevolume of CS-10 calculated to the same CS750 bag prepared above. Placeda red cap on the CS750 bag. Removed as much air as possible from theCS-750 bag as possible. Heat sealed the CS750 bag as close to the bag aspossible, removing the tubing. Label CS750 bag with “CRF Blank”, lotnumber, and initial/date. Placed the CRF Blank on cold packs until itwas placed in the CRF.

Calculated required volume of IL-2. Calculated the volume of IL-2 to addto the Final Product

TABLE 33 Calculated IL-2 volume Parameter Formula Result Final RetentateVolume Step 8.13.51 A. mL Final Formulated Volume B = A × 2 B. mL FinalIL-2 Concentration 300 IU/mL C. 300 IU/mL desired (IU/mL) IU of IL-2Required D = B × C D. IU IL-2 Working Stock from 6 × 10⁴ IU/mL E. 6 ×10⁴ IU/mL Step 8.11.33 Volume of IL-2 to Add to F = D ÷ E F. mL FinalProduct

Assembled Connect apparatus. Sterile welded a 4S-4M60 to a CC2 CellConnect replacing a single spike of the Cell Connect apparatus with the4-spike end of the 4S-4M60 manifold.

Assembled Connected apparatus. Sterile welded the CS750 Cryobags to theharness prepared above, replacing one of the four male luer ends (E)with each bag. Welded (per Process Note 5.11)CS-10 bags to spikes of the4S-4M60. Kept CS-10 cold by placing the bags between two cold packsconditioned at 2-8° C.

Prepared TIL with IL-2. Using an appropriately sized syringe, removedamount of IL-2 determined above from the “IL-2 6×104” aliquot. Connectthe syringe to the retentate bag prepared above via the Luer connectionand inject IL-2. Clear the line by pushing air from the syringe throughthe line.

Labeled Formulated TIL Bag. Closed the clamp on the transfer set andlabel bag as “Formulated TIL” and passed the bag out of the BSC.

Added the Formulated TIL bag to the apparatus. Once IL-2 had been added,welded the “Formulated TIL” bag to the remaining spike on the apparatus.

Added CS10. Passed the assembled apparatus with attached Formulated TIL,CS-750 bags, and CS-10 into the BSC. NOTE: The CS-10 bag and all CS-750bags were placed between two cold packs preconditioned at 2-8° C. Didnot place Formulated TIL bag on cold packs. Ensured all clamps wereclosed on the apparatus. Turn the stopcock so the syringe was closed.

Switched Syringes. Drew ˜10 mL of air into a 100 mL syringe and replacedthe 60 mL syringe on the apparatus.

Added CS10. Turned stopcock so that the line to the CS750 bags isclosed. Open clamps to the CS-10 bags and pull volume calculated aboveinto syringe. NOTE: Multiple syringes will be used to add appropriatevolume of CS-10. Closed clamps to CS-10 and open clamps to theFormulated TIL bag and add the CS-10. Add first 10.0 mL of CS10 atapproximately 10.0 mL/minute. Add remaining CS-10 at approximate rate of1.0 mL/sec. Note: Multiple syringes were used to add appropriate volumeof CS-10. Recorded time. NOTE: The target time from first addition ofCS-10 to beginning of freeze is 30 minutes. Recorded the volume of eachCS10 addition and the total volume added. Closed all clamps to the CS10bags.

Prepared CS-750 bags. Turned the stopcock so that the syringe was open.Opened clamps to the Formulated TIL bag and drew up suspension stoppingjust before the suspension reaches the stopcock. Closed clamps to theformulated TIL bag. Turned stopcock so that it was open to the emptyCS750 final product bags. Using a new syringe, removed as much air aspossible from the CS750 final product bags by drawing the air out. Whilemaintaining pressure on the syringe plunger, clamped the bags shut. Draw˜20 mL air into a new 100 mL syringe and connect to the apparatus. NOTE:Each CS-750 final product bag should be between two cold packs to keepformulated TIL suspension cold.

Dispensed cells. Turned the stopcock so the line to the final productbags was closed. Pulled the volume calculated above from the FormulatedTIL bag into the syringe. NOTE: Multiple syringes could be used toobtain correct volume. Turned the stopcock so the line to the formulatedTIL bag is closed. Working with one final product bag at a time,dispense cells into a final product bag. Recorded volume of cells addedto each CS750 bag above. Cleared the line with air from the syringe sothat the cells are even with the top of the spike port. Closed the clampon the filled bag. Repeated steps for each final product bag, gentlymixing formulated TIL bag between each. Recorded volume of TIL placed ineach final product bag below.

Removed air from final product bags and take retain. Once the last finalproduct bag was filled, closed all clamps. Drew 10 mL of air into a new100 mL syringe and replace the syringe on the apparatus. Manipulating asingle bag at a time, drew all of the air from each product bag plus thevolume of product for retain determined above. NOTE: Upon removal ofsample volume, inverted the syringe and used air to clear the line tothe top port of the product bag. Clamped the line to the bag once theretain volume and air was removed.

Recorded volume of retain removed from each bag.

Dispensed Retain. Dispensed retain into a 50 mL conical tube and labeltube as “Retain” and lot number. Repeat for each bag.

Prepared final product for cryopreservation. With a hemostat, clampedthe lines close to the bags. Removed syringe and red cap luer connectionon the apparatus that the syringe was on. Passed apparatus out of theBSC. Heat sealed (per Process Note 5.12) at F, removing the emptyretentate bag and the CS-10 bags. NOTE: Retained luer connection forsyringe on the apparatus. Disposed of empty retentate and CS-10 Bags.

Labeled final product bags. Attached sample final product label below.

Prepared final product for cryopreservation. Held the cryobags on coldpack or at 2-8° C. until cryopreservation.

Removed Cell Count Sample. Using an appropriately sized pipette, remove2.0 mL of retain removed above and placed in a 15 mL conical tube to beused for cell counts.

Performed Cell Counts. Performed cell counts and calculations utilizingthe NC-200. NOTE: Diluted only one sample to appropriate dilution toverify dilution is sufficient. Diluted additional samples to appropriatedilution factor and proceed with counts. Recorded Cell Count samplevolumes. NOTE: If no dilution needed, “Sample [μL]”=200, “Dilution[μL]”=0. Determined the Average of Viable Cell Concentration andViability of the cell counts performed.

Calculated Flow Cytometry Sample. Performed calculation to ensuresufficient cell concentration for flow cytometry sampling.

TABLE 34 Calculate flow cytometry cell concentration Viable Cell TargetVolume Required Concentration for 6 × 10⁷ TVC Is B ≤ 1.0 mL? A B = 6 ×10⁷ cells/A (Yes/No**) mL

Calculated IFN-γ. Sample Performed calculation to ensure sufficient cellconcentration for IFN-γ sampling.

Heat Sealed. Once sample volumes had been determined, heat sealed FinalProduct Bags as close to the bags as possible to remove from theapparatus.

TABLE 35 Labeling and collection of samples Sample Volume to Number ofAdd to Container Sample Containers Each Type *Mycoplasma 1 1.0 mL 15 mLConical Endotoxin 2 1.0 mL 2 mL Cryovial Gram Stain 1 1.0 mL 2 mLCryovial IFN-g 1 1.0 mL 2 mL Cryovial Flow 1 1.0 mL 2 mL CryovialCytometry **Bac-T 2 1.0 mL Bac-T Bottle Sterility QC Retain 4 1.0 mL 2mL Cryovial Satellite Vials 10 0.5 mL 2 mL Cryovial

For the Mycoplasma sample, add formulated cell suspension volume to the15 mL conical labelled “Mycoplasma Diluent” from above. Sterility &BacT. Testing Sampling. In the BSC, remove a 1.0 mL sample from theretained cell suspension collected in above using an appropriately sizedsyringe and inoculate the anaerobic bottle. Repeat the above for theaerobic bottle.

Labeled and stored samples. Labeled all samples with sample planinventory labels and store appropriately until transfer. Proceeded tonext steps for cryopreservation of final product and samples.

Final Product Cryopreservation

Prepared Controlled Rate Freezer. Verified the CRF had been set up priorto freeze. Record CRF Equipment. Cryopreservation is performed.

Set up CRF probes. Punctured the septum on the CRF blank bag. Insertedthe 6 mL vial temperature probe.

Placed final product and samples in CRF. Placed blank bag intopreconditioned cassette and transferred into the approximate middle ofthe CRF rack. Transferred final product cassettes into CRF rack andvials into CRF vial rack. Transferred product racks and vial racks intothe CRF. Recorded the time that the product is transferred into the CRFand the chamber temperature.

Determined the time needed to reach 4° C.±1.5° C. and proceed with theCRF run. Once the chamber temperature reached 4° C.±1.5° C., started therun. Recorded time.

Completed and Stored. Stopped the CRF after the completion of the run.Remove cassettes and vials from CRF. Transferred cassettes and vials tovapor phase LN2 for storage.

Example 12: Novel Cryopreserved Tumor Infiltrating Lymphocytes (LN-144)Administered to Patients with Metastatic Melanoma Demonstrated Efficacyand Tolerability in a Multicenter Phase 2 Clinical Trial Background

The safety and efficacy of adoptive cell therapy (ACT) utilizing tumorinfiltrating lymphocytes (TIL) has been studied in hundreds of patientswith metastatic melanoma, and has demonstrated meaningful and durableobjective response rates (ORR).¹ In an ongoing Phase 2 trial, C-144-01utilizing centralized GMP manufacturing of TIL, both non-cryopreservedGeneration 1 (Gen 1) and cryopreserved Generation 2 (Gen 2) TILmanufacturing processes were assessed.

Gen 1 is approximately 5-6 weeks in duration of manufacturing(administered in Cohort 1 of C-144-01 study), while Gen 2 is 22 days induration of manufacturing (process 2A, administered in Cohort 2 ofC-144-01 study). Preliminary data from Cohort 1 patients infused withthe Gen 1 LN-144 manufactured product, was encouraging in treatingpost-PD-1 metastatic melanoma patients as the TIL therapy producedresponses.² Benefits of Gen 2 included: (A) reduction in the timepatients and physicians wait to infuse TIL to patient; (B)cryopreservation permits flexibility in scheduling, distribution, anddelivery; and (C) reduction of manufacturing costs. Preliminary datafrom Cohort 2 is presented herein. FIG. 25 shows an embodiment of theGen 2 cryopreserved LN-144 manufacturing process (process 2A).

Study Design: C-144-01 Phase 2 Trial in Metastatic Melanoma

Phase 2, Multicenter, 3-Cohort Study to Assess the Efficacy and Safetyof Autologous Tumor Infiltrating Lymphocytes (LN-144) for Treatment ofPatients with Metastatic Melanoma.

Key Inclusion Criteria: (1) Measurable metastatic melanoma and ≥1 lesionresectable for TIL generation; (2) Progression on at least one priorline of systemic therapy; (3) Age ≥18; and (4) ECOG PS 0-1.

Treatment Cohorts: (1) Non-Cryopreserved LN-144 product; (2)Cryopreserved LN-144 product; and (3) Retreatment with LN-144 forpatients without response or who progress after initial response. FIG.26 shows the study design.

Endpoints: (1) Primary: Efficacy defined as ORR and (2) Secondary:Safety and Efficacy.

Methods

Cohort 2 Safety Set: 13 patients who underwent resection for the purposeof TIL generation and received any component of the study treatment.

Cohort 2 Efficacy Set: 9 patients who received the NMA-LDpreconditioning, LN-144 infusion and at least one dose of IL-2, and hadat least one efficacy assessment. 4 patients did not have an efficacyassessment at the time of the data cut.

Biomarker data has been shown for all available data read by the date ofthe data cut.

Results

FIG. 27 provides a table illustrating the Comparison PatientCharacteristics from Cohort 1 (ASCO 2017) vs Cohort 2. Cohort 2 has: 4median prior therapies; all patients have received prior anti-PD-1 andanti-CTLA-4; and had higher tumor burden reflected by greater sum ofdiameters (SOD) for target lesions and higher mean LDH at Baseline. FIG.28 provides a table showing treatment emergent adverse events (≥30%).

For Cohort 2 (cryopreserved LN-144), the infusion product and TILtherapy characteristics were (1) mean number of TIL cells infused:37×10⁹, and (2) median number of IL-2 doses administrations was 4.5.FIG. 29 shows the efficacy of the infusion product and TIL therapy forPatients #1 to #8.

FIG. 30 shows the clinical status of response evaluable patients withstable disease (SD) or a better response. A partial response (PR) forPatient 6 was unconfirmed as the patient did not reached the secondefficacy assessment yet. One patient (Patient 9) passed away prior tothe first assessment (still considered in the efficacy set).

Of the 9 patients in the efficacy set, one patient (Patient 9) was notevaluable (NE) due to melanoma-related death prior to first tumorassessment not represented on FIG. 30. Responses were seen in patientstreated with Gen 2. The disease control rate (DCR) was 78%. Time toresponse was similar to Cohort 1. One patient (Patient 3) withprogressive disease (PD) as best response was not included in the swimlane plot.

FIG. 31 shows the percent change in sum of diameters. Patient 9 had nopost-LN-144 disease assessment due to melanoma-related death prior toDay 42. Day −14: % change of Sum of Diameters from Screening to Baseline(Day −14). Day −14 to Day 126: % change of SOD from Baseline. Day−14=Baseline. Day 0=LN-144 infusion.

Upon TIL treatment, an increase of HMGB1 was observed (FIG. 32). PlasmaHMGB1 levels were measured using HMGB1 ELISA kit (Tecan US, Inc). Datashown represents fold change in HMGB1 levels pre (Day −7) and post (Day4 and Day 14) LN-144 infusion in Cohort 1 and Cohort 2 patients (pvalues were calculated using two-tailed paired t-test based onlog-transformed data). Sample size (bold and italicized) and mean(italicized) values are shown in parentheses for each time point. HMGB1is secreted by activated immune cells and released by damaged tumorcells. The increased HMGB1 levels observed after treatment with LN-144are therefore suggestive of an immune-mediated mechanism of anti-tumoractivity.

Plasma IP-10 levels were measured using Luminex assay. Data shown inFIG. 33 represents fold change in IP-10 levels pre (Day −7) and post(Day 4 and Day 14) LN-144 infusion in Cohort 1 and Cohort 2 patients (pvalues were calculated using two-tailed paired t-test based onlog-transformed data). Sample size (bold and italicized) and mean(italicized) values are shown in parentheses for each time point. Thepost-LN-144 infusion increase in IP-10 is being monitored to understandpossible correlation with TIL persistence.

Updated data from Cohort 2 (n=17 patients) is reported in FIG. 34 toFIG. 39. In comparison to Cohort 1 and an embodiment of the Gen 1process, which showed a DCR of 64% and an overall response rate (ORR) of29% (N=14), Cohort 2 and an embodiment of the Gen 2 process showed a DCRof 80% and an ORR of 40% (N=10).

CONCLUSIONS

Preliminary results from the existing data demonstrate comparable safetybetween Gen 1 and Gen 2 LN-144 TIL products. Administration of TILsmanufactured with the Gen 2 process (process 2A, as described herein)leads to surprisingly increased clinical responses seen in advanceddisease metastatic melanoma patients, all had progressed on anti-PD-1and anti-CTLA-4 prior therapies. The DCR for cohort 2 was 78%.

Preliminary biomarker data is supportive of the cytolytic mechanism ofaction proposed for TIL therapy.

The embodiment of the Gen 2 manufacturing process described herein takes22 days. This process significantly shortens the duration of time apatient has to wait to receive their TIL, offers flexibility in thetiming of dosing the patients, and leads to a reduction of cost ofmanufacturing, while providing other advantages over prior approachesthat allow for commercialization and registration with health regulatoryagencies. Preliminary clinical data in metastatic melanoma using anembodiment of the Gen 2 manufacturing process also indicates asurprising improvement in clinical efficacy of the TILs, as measured byDCR, ORR, and other clinical responses, with a similar time to responseand safety profile compared to TILs manufactured using the Gen 1process.

REFERENCES

-   ¹Goff, et al. Randomized, Prospective Evaluation Comparing Intensity    of Lymphodepletion Before Adoptive Transfer of Tumor-Infiltrating    Lymphocytes for Patients With Metastatic Melanoma. J Clin Oncol.    2016 Jul. 10; 34(20):2389-97.-   ²Sarnaik A, Kluger H, Chesney J, et al. Efficacy of single    administration of tumor-infiltrating lymphocytes (TIL) in heavily    pretreated patients with metastatic melanoma following checkpoint    therapy. J Clin Oncol. 2017; 35 [suppl; abstr 3045].

Example 13: Historical Control Study

A historical control study may be used for comparison of the treatmentoutcomes in patients with double-refractory metastatic melanoma to theoutcomes of TIL therapies disclosed herein, such as those therapiesdescribed in Example 2 and Example 3. In an embodiment, a patienttreated with TIL therapies disclosed herein exhibits an improvedresponse to the response expected from a historical control. In anembodiment, a patient treated with TIL therapies disclosed hereinexhibits an improved response to the response expected from a historicalcontrol, wherein the improved response is determined as overall responserate. In an embodiment, a patient treated with TIL therapies disclosedherein exhibits an improved response to the response expected from ahistorical control, wherein the improved response is determined asoverall response rate, wherein the improvement in overall response rateis at least 5%, at least 10%, at least 15%, at least 20%, at least 25%,or at least 50%. In an embodiment, a patient treated with TIL therapiesdisclosed herein exhibits an improved response to the response expectedfrom a historical control, wherein the improved response is determinedas duration of response. In an embodiment, a patient treated with TILtherapies disclosed herein exhibits an improved response to the responseexpected from a historical control, wherein the improved response isdetermined as duration of response, wherein the improvement in durationof response is at least 5%, at least 10%, at least 15%, at least 20%, atleast 25%, or at least 50%.

The historical control study to determine the response ofdouble-refractory patients to other therapies may be performed usingdata obtained from the treatment records of metastatic melanomapatients. Patients must be exposed to 3 or more lines of therapies formelanoma after the initial diagnosis of melanoma. Lines of systemictherapies are counted per the following rules and start and stop datesof each therapy are considered:

-   -   All agents received within 28 days of the first line (1 L) start        constituted the 1 L regimen, and could include a single agent or        combine multiple agents; 1 L end corresponded to either the        first gap of >90 days in the all 1 L agents or the initiation of        a new agent that was not part of 1 L (i.e., switch to a new        line);    -   Subsequent lines of therapy are identified as the earliest        of (a) initiation of an agent not in the previous treatment        regimen (after the initial 28 day period to identify 1 L        regimen), or (b) initiation of any agent after a gap of >90 days        in the previous treatment. Regimens used in subsequent lines        were identified based on all agents received within 28 days of        the start of the respective line of therapy    -   In general, ipilumimab and nivolumab administered as a        combination are considered to be one therapy and BRAF and MEK        inhibitors administered in combination (e.g., dabrafenib and        trametinib) considered to be one therapy.    -   All patients must have been treated with at least one anti-PD-1        (or anti-PD-L1) therapy and failed (i.e., are refractory or        relapsed). Availability of the scan date that led to disease        progression if the line of therapy contains anti-PD 1 therapy is        preferred. For the last therapy on record (3rd or later), an        overall response per visit and date of either disease        progression or death (if applicable) are required.    -   For the last line of therapy on record, either i) target and        non-target lesions measures per each assessment, or ii) overall        response per visit by RECIST is required.    -   Certain baseline disease status and baseline characteristics        before the initiation of the last therapy on record are        required, to allow for evaluation of whether these patients meet        the similar eligibility criteria for other studies described        herein (including in Example 2 and Example 3).

Example 14: Safety and Efficacy of Cryopreserved Autologous TumorInfiltrating Lymphocyte Therapy (LN-144, Lifileucel) in AdvancedMetastatic Melanoma Patients Following Progression on CheckpointInhibitors

Adoptive cell therapy (ACT) utilizing tumor-infiltrating lymphocytes(TIL) leverages and enhances the body's natural defense against cancer.TIL has demonstrated antitumor efficacy. Durable long-term responses inheavily pretreated patients (Rosenberg, S. A., et al. Durable CompleteResponses in Heavily Pretreated Patients with Metastatic Melanoma UsingT-Cell Transfer Immunotherapy. Clinical Cancer Research, 17(13),4550-4557).

C-144-01 (NCT02360579) is an ongoing Phase 2 multicenter study, focusingon autologous TIL (lifileucel; LN-144). Patient population being focusedon is unresectable metastatic melanoma who have progressed on checkpointinhibitors and BRAF/MEK inhibitors (if BRAF mutated). TIL manufacturingconditions: central manufacturing of cryopreserved TIL, 22 day duration(based on the processes described herein).

C-144-01 Phase 2 Trial in Metastatic Melanoma:

Phase 2, multicenter study to assess the efficacy and safety ofautologous Tumor Infiltrating Lymphocytes (LN-144) for treatment ofpatients with metastatic melanoma (NCT02360579)

Endpoints of the Study:

Primary: Efficacy defined as investigator assessed ORR. Secondary:Safety and efficacy Study Details:

Cohort 2 fully enrolled. Cohort 2 preliminary efficacy, safety andbiomarker data provided in FIGS. 40-48 herein (n=47).

Cohort 4 will initiate in the future with 80-100 patients.

Methods:

Data for Cohort 2.

Cohort 2 Safety & Efficacy Sets: 47 patients who underwent resection forthe purpose of TIL generation and received lifileucel infusion.Biomarker data has been shown for all available data read by the date ofthe data cut.

IP-10 is measured by a commercial Bio-Rad bead-based Bio-Pleximmunoassay, which measures multiple cytokines and chemokines, and whichincludes an antibody specific for IP-10.

IP-10 is measured by taking blood samples from the patient and ismeasured in the plasma fraction obtained from the blood (i.e., after allblood cells are removed) and is reported in units of picograms permilliliter of plasma (i.e., pg/mL).

Cohort 2 (Lifileucel): Infusion Product and TIL Therapy Characteristics

Mean number of TIL cells infused: 26×10⁹. Median number of IL-2 dosesadministered was 6.0. Overall, 72% of patients had a reduction in tumorburden. Median follow up is 6.0 months. Median duration of response(DOR) is 6.4 months. Range of DOR was from 1.3+ to 14+ months. Change inIP-10 levels in periphery may have a correlation with response.

Mean change in IP-10 levels from baseline to day 1 post TIL infusion washigher among responders vs. nonresponders (p=0.19)

In heavily pretreated metastatic melanoma patients, efficacy to date isnotable:

-   -   Overall response rate (ORR): 38%    -   Median DOR: 6.4 months, range 1.3+ to 14+    -   Disease control rate (DCR): 77%    -   16/17 had no response to prior anti-PD-1

The range of IP-10 in responders and non responders is provided inTables 1 and 2 below. These recite the actual numbers, in pg/mL.Negative values mean decrease in IP-10 from day −7 to day 1 wasobserved.

Except the 4 patients in responders, remaining of responders (7responder patients) have change in IP-10 level value more than 1656pg/ml.

TABLE 1 Change in levels of IP-10 from Day −7 to Day 1 (Range in pg/ml)Minimum Maximum Responders −264 9711.84 NonResponders −9808 4902.9

TABLE 2 Responders Non Responders 9711.84 932.23 2880.3 906.45 3105.92−44.17 4923.37 4902.9 3960.7 1113.27 −65.99 2210.6 −264.25 263.81 313.38166.41 2121.34 1236.29 1656.62 738.03 −72.48 −237.04 937.84 3109.53308.36 3760.47 −9808.01 1082.79 868.95 −337.47 −27.29 287 1667.5 313.73437.59

Biomarker analyses show that an increase in IP-10 levels may correlatewith anti-tumor response.

Preliminary data supports lifileucel (also known as LN-144) autologousTIL as an efficacious and well-tolerated therapeutic option for patientswith metastatic melanoma.

1. A method of treating double-refractory metastatic melanoma in apatient in need thereof, the method comprising administering atherapeutically effective population of tumor infiltrating lymphocytes(TILs) to the patient.
 2. The method of claim 1, wherein thedouble-refractory metastatic melanoma is a cutaneous double-refractorymetastatic melanoma.
 3. The method of claim 1, wherein thedouble-refractory metastatic melanoma is refractory to at least twoprior systemic treatment courses, not including neo-adjuvant or adjuvanttherapies.
 4. The method of claim 1, wherein the double-refractorymetastatic melanoma is refractory to aldesleukin or a biosimilarthereof.
 5. The method of claim 1, wherein the double-refractorymetastatic melanoma is refractory to pembrolizumab or a biosimilarthereof.
 6. The method of claim 1, wherein the double-refractorymetastatic melanoma is refractory to nivolumab or a biosimilar thereof.7. The method of claim 1, wherein the double-refractory metastaticmelanoma is refractory to ipilimumab or a biosimilar thereof.
 8. Themethod of claim 1, wherein the double-refractory metastatic melanoma isrefractory to ipilimumab or a biosimilar thereof and pembrolizumab or abiosimilar thereof.
 9. The method of claim 1, wherein thedouble-refractory metastatic melanoma is refractory to ipilimumab or abiosimilar thereof and nivolumab or a biosimilar thereof.
 10. The methodof claim 1, wherein the double-refractory metastatic melanoma isrefractory to a BRAF inhibitor.
 11. The method of claim 1, wherein thedouble-refractory metastatic melanoma is refractory to a PD-L1inhibitor.
 12. The method of claim 11, wherein the PD-L1 inhibitor isselected from the group consisting of avelumab, atezolizumab,durvalumab, and biosimilars thereof.
 13. The method of claim 1, whereinthe double-refractory metastatic melanoma is refractory to a combinationof a PD-1 inhibitor and a CTLA-4 inhibitor.
 14. The method of claim 13,wherein the PD-1 inhibitor is nivolumab or a biosimilar thereof and theCTLA-4 inhibitor is selected from the group consisting of ipilumumab,tremelimumab, and biosimilars thereof.
 15. The method of claim 1,wherein the double-refractory metastatic melanoma is refractory to acombination of a BRAF inhibitor and a MEK inhibitor.
 16. The method ofclaim 15, wherein the BRAF inhibitor is dabrafenib or apharmaceutically-acceptable salt thereof and the MEK inhibitor istrametinib or a pharmaceutically-acceptable salt or solvate thereof. 17.The method of claim 1, wherein the metastatic melanoma is resistant to aPD-1 inhibitor or PD-L1 inhibitor.
 18. The method of claim 17, whereinthe PD-1 or PD-L1 inhibitor is selected from the group consisting ofnivolumab, pembrolizumab, avelumab, atezolizumab, durvalumab, andbiosimilars thereof.
 19. The method of claim 1, wherein the patient doesnot possess a BRAF mutation.
 20. The method of claim 1, wherein thepatient has received at most 4 doses of nivolumab or a biosimilarthereof prior to receiving the therapeutically effective population ofTILs.
 21. The method of claim 1, wherein the patient has progressed orhad no response to at least two prior systemic treatment courses. 22.The method of claim 1, wherein the patient exhibits an increase in thelevel of IP-10 after administration of the therapeutically effectivepopulation of tumor infiltrating lymphocytes (TILs).
 23. The method ofclaim 22, wherein the increase in the level of IP-10 is indicative oftreatment response and/or treatment efficacy.
 24. The method of claim22, wherein the patient is administered one or more further dosages of atherapeutically effective population of tumor infiltrating lymphocytes(TILs).
 25. The method of claim 22, wherein the patient is notadministered a further dosage of a therapeutically effective populationof tumor infiltrating lymphocytes (TILs).
 26. A method of treatingdouble-refractory metastatic melanoma in a patient in need thereof, themethod comprising: (a) obtaining a first population of TILs from a tumorresected from the patient by processing a tumor sample obtained from thepatient into multiple tumor fragments; (b) adding the tumor fragmentsinto a closed system; (c) performing a first expansion by culturing thefirst population of TILs in a cell culture medium comprising IL-2, andoptionally OKT-3, to produce a second population of TILs, wherein thefirst expansion is performed in a closed container providing a firstgas-permeable surface area, wherein the first expansion is performed forabout 3-14 days to obtain the second population of TILs, wherein thesecond population of TILs is at least 50-fold greater in number than thefirst population of TILs, and wherein the transition from step (b) tostep (c) occurs without opening the system; (d) performing a secondexpansion by supplementing the cell culture medium of the secondpopulation of TILs with additional IL-2, optionally OKT-3, and antigenpresenting cells (APCs), to produce a third population of TILs, whereinthe second expansion is performed for about 7-14 days to obtain thethird population of TILs, wherein the third population of TILs is atherapeutic population of TILs, wherein the second expansion isperformed in a closed container providing a second gas-permeable surfacearea, and wherein the transition from step (c) to step (d) occurswithout opening the system; (e) harvesting the therapeutic population ofTILs obtained from step (d) to provide a harvested TIL population,wherein the transition from step (d) to step (e) occurs without openingthe system; (f) transferring the harvested TIL population from step (e)to an infusion bag, wherein the transfer from step (e) to (f) occurswithout opening the system, and optionally cryopreserving the harvestedTIL population and (g) administering a therapeutically effective amountof the harvested TIL population to the patient with double-refractorymetastatic melanoma.
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 45. The method of claim 26, wherein the IL-2 is presentat an initial concentration of between 1000 IU/mL and 6000 IU/mL in thecell culture medium in the first expansion step (c).
 46. The method ofclaim 26, wherein in the second expansion step (d), the IL-2 is presentat an initial concentration of between 1000 IU/mL and 6000 IU/mL and theOKT-3 antibody is present at an initial concentration of about 30 ng/mL.47. (canceled)
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 61. A method of treating double-refractory metastaticmelanoma in a patient in need thereof, the method comprising: (a)obtaining a first population of TILs from a tumor resected from thepatient by processing a tumor sample obtained from the patient intomultiple tumor fragments; (b) adding the tumor fragments into a closedsystem; (c) performing a first expansion by culturing the firstpopulation of TILs in a cell culture medium comprising IL-2, andoptionally OKT-3, to produce a second population of TILs, wherein thefirst expansion is performed in a closed container providing a firstgas-permeable surface area, wherein the first expansion is performed forabout 3-14 days to obtain the second population of TILs, wherein thesecond population of TILs is at least 50-fold greater in number than thefirst population of TILs, and wherein the transition from step (b) tostep (c) occurs without opening the system; (d) performing a secondexpansion by supplementing the cell culture medium of the secondpopulation of TILs with additional IL-2, optionally OKT-3, and antigenpresenting cells (APCs), to produce a third population of TILs, whereinthe second expansion is performed for about 7-14 days to obtain thethird population of TILs, wherein the third population of TILs is atherapeutic population of TILs, wherein the second expansion isperformed in a closed container providing a second gas-permeable surfacearea, and wherein the transition from step (c) to step (d) occurswithout opening the system; (e) harvesting the therapeutic population ofTILs obtained from step (d) to provide a harvested TIL population,wherein the transition from step (d) to step (e) occurs without openingthe system; (f) transferring the harvested TIL population from step (e)to an infusion bag, wherein the transfer from step (e) to (f) occurswithout opening the system, and optionally cryopreserving the harvestedTIL population; (g) administering a therapeutically effective amount ofthe harvested TIL population to the patient with double-refractorymetastatic melanoma; and (h) measuring the level of IP-10 in the patientafter administering a therapeutically effective amount of the TILs instep (g).
 62. A method of treating cancer in a patient in need thereof,the method comprising: (a) obtaining a first population of TILs from atumor resected from the patient by processing a tumor sample obtainedfrom the patient into multiple tumor fragments; (b) adding the tumorfragments into a closed system; (c) performing a first expansion byculturing the first population of TILs in a cell culture mediumcomprising IL-2, and optionally OKT-3, to produce a second population ofTILs, wherein the first expansion is performed in a closed containerproviding a first gas-permeable surface area, wherein the firstexpansion is performed for about 3-14 days to obtain the secondpopulation of TILs, wherein the second population of TILs is at least50-fold greater in number than the first population of TILs, and whereinthe transition from step (b) to step (c) occurs without opening thesystem; (d) performing a second expansion by supplementing the cellculture medium of the second population of TILs with additional IL-2,optionally OKT-3, and antigen presenting cells (APCs), to produce athird population of TILs, wherein the second expansion is performed forabout 7-14 days to obtain the third population of TILs, wherein thethird population of TILs is a therapeutic population of TILs, whereinthe second expansion is performed in a closed container providing asecond gas-permeable surface area, and wherein the transition from step(c) to step (d) occurs without opening the system; (e) harvesting thetherapeutic population of TILs obtained from step (d) to provide aharvested TIL population, wherein the transition from step (d) to step(e) occurs without opening the system; (f) transferring the harvestedTIL population from step (e) to an infusion bag, wherein the transferfrom step (e) to (f) occurs without opening the system, and optionallycryopreserving the harvested TIL population; (g) administering atherapeutically effective amount of the harvested TIL population to thepatient with double-refractory metastatic melanoma; and (h) measuringthe level of IP-10 in the patient after administering a therapeuticallyeffective amount of the TILs in step (g).
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 78. A methodof predicting a treatment response and/or predicting treatment efficacyfor administration of a therapeutically effective amount of tumorinfiltrating lymphocytes (TILs) to a patient, the method comprising: a)obtaining a biological sample from a patient with cancer, includingdouble-refractory metastatic melanoma; b) measuring the level of IP-10in the biological sample from a); c) administering a therapeuticallyeffective amount of TILs; d) obtaining a biological sample from thepatient after the administration of the therapeutically effective amountof TILs in step c) e) measuring the level of IP-10 in the biologicalsample from d); f) predicting a treatment response to and/or predictingtreatment efficacy of the administration of the therapeuticallyeffective amount of the TILs based upon the level of IP-10 measuredafter administration as compared to the level of IP-10 measured prior toadministration.
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